Soluble endoglin and uses thereof

ABSTRACT

The invention provides isolated soluble endoglin polypeptides, nucleic acids encoding soluble endoglin polypeptides, antibodies that specifically bind soluble endoglin polypeptides, and kits containing these materials. The invention also provides methods for treating or decreasing the likelihood of developing a soluble endoglin-mediated disorder in a subject requiring the administration of an agent capable of reducing the expression or biological activity of a soluble endoglin polypeptide and methods for treating or decreasing the likelihood of developing a soluble endoglin-preventive disorder in a subject requiring the administration of a soluble endoglin polypeptide or a nucleic acid encoding the soluble endoglin polypeptide. The invention further provides methods for the diagnosis of a soluble endoglin-mediated disorder or a soluble endoglin-preventive disorder and methods for identifying a compound to treat a soluble endoglin-mediated or a soluble endoglin-preventive disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.provisional application No. 61/294,177, filed Jan. 12, 2010, hereinincorporated by reference.

FIELD OF THE INVENTION

In general, this invention relates to soluble endoglin polypeptides andnucleic acids, and uses of soluble endoglin polypeptides and nucleicacids or soluble endoglin-specific compounds.

BACKGROUND OF THE INVENTION

Endoglin, also known as CD105, is a homodimeric cell membraneglycoprotein that is predominantly expressed on endothelial cells suchas syncytiotrophoblasts, human umbilical vein endothelial cells (HUVEC),and on vascular endothelial cells. Endoglin shares sequence identitywith betaglycan, a transforming growth factor (TGF)-β receptor type III.Endoglin has been shown to be a regulatory component of the TGF-βreceptor complex, which modulates angiogenesis, proliferation,differentiation, and apoptosis. Endoglin also binds several othermembers of the TGF-β superfamily including activin-A, bone morphogenicprotein (BMP)-2, and BMP-7. In particular, endoglin binds TGF-β1 andTGF-β3 with high affinity and forms heterotrimeric associations with theTGF-β signaling receptors types I and II. Mutations in the coding regionof the endoglin gene are responsible for haemorrhagic telangiectasiatype 1 (HHT1), a dominantly inherited vascular disorder characterized bymultisystemic vascular dysplasia and recurrent hemorrhage.

A soluble form of endoglin was previously identified and found tointerfere with TGF-β1 signaling and endothelial nitric oxide synthase(eNOS) activation in endothelial cells, thereby disrupting mechanismsnecessary for maintenance of vascular health. Soluble endoglin was foundto be present at increased levels in patients with metastatic breast andcolorectal cancer. It has also been shown that patients havingpre-eclampsia produce large quantities of soluble endoglin and thatsoluble endoglin contributes to the pathogenesis of pre-eclampsia.

There is a need for methods of treating and diagnosing subjects at riskfor or having a soluble endoglin-mediated disorder (e.g., eclampsia andpre-eclampsia) or a soluble endoglin-preventive disorder (e.g.,disorders characterized by increased TGF β expression or biologicalactivity).

SUMMARY OF THE INVENTION

We have discovered a form of soluble endoglin produced by alternativesplicing of the endoglin precursor mRNA. We have isolated thispreviously unrecognized mRNA transcript of endoglin from human tissueand discovered that it encodes a stable and soluble (secreted) protein.This soluble form of endoglin exists predominantly as a monomer and isable to modulate TGF-β/BMP signaling. The soluble endoglin describedherein includes a unique C-terminal amino acid sequence of amino acids437-444 of RVRWTVTC (SEQ ID NO: 1) or amino acids 438-444 of VRWTVTC(SEQ ID NO: 2) and has a soluble endoglin biological activity. Theunique C-terminal amino acid sequences and the complete amino acid andnucleic acid sequences of the alternatively spliced mRNA transcript ofsoluble endoglin are shown in FIGS. 2A, 3B, and 1, respectively.

The unique amino acid sequences incorporated into soluble endoglin as aresult of alternative splicing can be used to generate solubleendoglin-specific compounds (e.g., an antibody, a small moleculeinhibitor, or a small inhibitory RNA, such as miRNA or siRNA) thatspecifically target, bind, or modulate the expression or activity ofthis form of soluble endoglin. For example, antibodies can be used tospecifically detect or modulate the biological activity or expression ofthis protein in a number of soluble endoglin-mediated diseases includingbut not limited to pregnancy related hypertensive disorders (e.g.,pre-eclampsia, eclampsia, gestational hypertension, chronichypertension, HELLP syndrome, and pregnancy with a SGA infant),pulmonary hypertension, and malaria. In addition, elevated solubleendoglin levels have been detected in cancers, in senior patients withAlzheimer's disease and in patients with cerebral arteriovenousmalformations, and individuals with suspected left ventriculardysfunction also display elevated soluble endoglin levels suggestingthat circulating levels of this protein may be a sensitive measure ofelevated left heart filling pressures. Moreover, soluble endoglin levelsare elevated in patients with diabetes having retinopathy and/or a highprobability of 10-year cardiovascular risk, as well as in patients withdiabetes and hypertension who have three or more damaged target organs(e.g., heart, vessels, and kidney). Compounds that reduce, decrease, orinhibit the expression levels or biological activity of soluble endoglincan be used to treat any of the above endoglin-mediated diseases.

Alternatively, isolated soluble endoglin polypeptides and fragments,derivatives, or analogs thereof, and agents that increase the expressionor a biological activity of a soluble endoglin polypeptide of theinvention may be used to treat a subject having a solubleendoglin-preventive disorder such as those characterized by factors(e.g. increased TGF-β levels or activity) or events (e.g. fibrosis,angiogenesis, immune activation) that contribute to the disorder and/orthat can be inhibited or modulated by soluble endoglin. Examples includefibrotic disorders of internal organs and the scarring of skin whereTGF-β is a significant contributor, disorders characterized by excessiveangiogenesis (e.g. hemangiomas, pulmonary capillary hemangiomatosis) orabnormal growth of blood vessel such as cancer (e.g., cancer of thebreast, prostate, colon, lung, head and neck, liver, kidney, renalsystem, and endometrium) and inflammatory and immune disorders.

Accordingly, in a first aspect, the invention features an isolatedsoluble endoglin polypeptide that includes a sequence that is at least85% identical to VRWTVTC (SEQ ID NO: 2) and has a soluble endoglinbiological activity. In one embodiment, the sequence that is at least85% identical to SEQ ID NO: 2 is located at the C-terminus of thesoluble endoglin polypeptide. In another embodiment the soluble endoglinpolypeptide includes the sequence of SEQ ID NO: 2, desirably at theC-terminus of the soluble endoglin.

In various embodiments, the isolated soluble endoglin polypeptideincludes or consists of an amino acid sequence that is at least 98%,99%, or 100% identical to SEQ ID NO: 3. The soluble endoglin polypeptidecan include fragments of soluble endoglin, for example, fragments thatinclude a total of at least 100, 200, 250, 300, 350, 400, or 444 aminoacids. The soluble endoglin polypeptide, or fragments thereof, canfurther include an additional amino acid sequence at the N- orC-terminus, which can be at least 5, 10, 15, 20, 25 amino acids or more.

In various embodiments, the soluble endoglin polypeptide biologicalactivity is selected from the group consisting of: the ability to bindto TGF-β1, the ability to bind to TGF-β3, the ability to bind toactivin-A, the ability to bind to bone morphogenic protein(BMP)-2, theability to bind to BMP-7, the ability to bind to BMP-9, the ability tobind to BMP receptor II, the ability to bind TGF-β receptor I, theability to bind to TGF-β receptor II, the ability to reverse or inhibitangiogenesis, the ability to reduce or inhibit Smad 2/3-dependenttranscriptional activation, and the ability to inhibit endothelialnitric oxide synthase activation.

The isolated soluble endoglin polypeptide can further include one ormore modifications selected from the group consisting of: acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, S-nitrosylation, disulfide bond formation,demethylation, formation of cysteine, formation of sulfonic, sulfenic,or sulfinic acid, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids, ubiquitination,D-isomerization, derivitization of water soluble polymers, addition of adetectable label, and conjugation of another protein or therapeuticcompound. In one embodiment, the modification includes glycosylation,oxidation, or s-nitrosylation.

In another embodiment, the invention features an antibody, or antibodybinding fragment thereof, that specifically binds to a soluble endoglinpolypeptide, where the antibody, or antibody-binding fragment, binds toan epitope comprising a sequence that is at least 85% identical toVRWTVTC (SEQ ID NO: 2), or a fragment thereof. Desirably, the epitopefragment is at least 3 amino acids, 4 amino acids, 5 amino acids, or 6amino acids. In one embodiment, the antibody or antibody-bindingfragment binds to an epitope that includes SEQ ID NO: 2. In anotherembodiment, the antibody or antibody-binding fragment competes with theantibody C-5144 for binding to soluble endoglin.

In various embodiments, the antibody, or antibody binding fragmentinhibits one of more of the following activities of soluble endoglin:the ability to bind to TGF-β1, the ability to bind to TGF-β3, theability to bind to activin-A, the ability to bind to bone morphogenicprotein(BMP)-2, the ability to bind to BMP-7, the ability to bind toBMP-9, the ability to bind to BMP receptor II, the ability to bind TGF-βreceptor I, the ability to bind to TGF-β receptor II, the ability toreverse or inhibit angiogenesis, the ability to reduce or inhibit Smad2/3-dependent transcriptional activation, and the ability to inhibitendothelial nitric oxide synthase activation.

The antibody, or fragment thereof, can be a monoclonal antibody, achimeric, a humanized, a human antibody, an antibody that lacks an Fcportion or is a F(ab′)₂, a Fab, or an Fv structure.

In various embodiments, the antibody or antibody binding fragment is ina pharmaceutically acceptable carrier.

In another embodiment, the invention features an isolated nucleic acidmolecule that includes a sequence that is at least 95% identical to thesequence of SEQ ID NO: 4 and that includes a sequence that encodes anamino acid sequence that has a soluble endoglin biological activitywherein the amino acid sequence includes a sequence that is at least 85%identical to VRWTVTC (SEQ NO: 2).

In addition embodiments, the sequence that encodes an amino acidsequence that is at least 85% identical to SEQ ID NO: 2 is located atthe 3′ terminus of the nucleic acid molecule. In various embodiments,the nucleic acid includes a sequence that encodes the amino acidsequence of SEQ ID NO: 2.

In another aspect, the invention features an isolated inhibitory nucleicacid molecule, that includes at least one strand that is complementaryto a nucleic acid sequence that encodes an amino acid sequence that isat least 85% identical to VRWTVTC (SEQ ID NO: 2), or a fragment thereof,and wherein the inhibitory nucleic acid molecule reduces or inhibits theexpression or a biological activity of a soluble endoglin polypeptide.In one embodiment, the inhibitory nucleic acid molecule has at least onestrand that is complementary to a nucleic acid sequence that encodes theamino acid sequence of SEQ ID NO: 2. In another embodiment, the isolatedinhibitory nucleic acid molecule reduces or inhibits a biologicalactivity of a soluble endoglin that is selected from the groupconsisting of: the ability to bind to TGF-β1, the ability to bind toTGF-β3, the ability to bind to activin-A, the ability to bind to bonemorphogenic protein(BMP)-2, the ability to bind to BMP-7, the ability tobind to BMP-9, the ability to bind to BMP receptor II, the ability tobind TGF-β receptor I, the ability to bind to TGF-β receptor II, theability to reverse or inhibit angiogenesis, the ability to reduce orinhibit Smad 2/3-dependent transcriptional activation, and the abilityto inhibit endothelial nitric oxide synthase activation.

In various embodiments, the isolated inhibitory nucleic acid molecule isa double-stranded RNA, a small interfering RNA (siRNA) or a micro RNA(miRNA). The siRNA can be between 17 to 25 nucleotides in length. Inanother embodiment, the isolated inhibitor nucleic acid molecule issingle-stranded DNA or an antisense nucleobase oligomer.

In another aspect, the invention features a method of treating ordecreasing the likelihood of developing a soluble endoglin-mediateddisorder in a subject, that includes the step of administering to thesubject an agent capable of reducing the expression or biologicalactivity of a soluble endoglin polypeptide, wherein the soluble endoglinpolypeptide comprises a sequence that is at least 85% identical toVRWTVTC (SEQ ID NO: 2). In one embodiment, the administering results ina reduction of the expression or biological activity of a solubleendoglin polypeptide that includes the sequence of SEQ ID NO: 2. Inanother embodiment, the administering results in a reduction of theexpression or biological activity of a soluble endoglin polypeptide thatincludes an amino acid sequence that is at least 98% identical to thecorresponding amino acid sequence in SEQ ID NO: 3. The biologicalactivity of the soluble endoglin polypeptide can include the ability tobind to TGF-β1, the ability to bind to TGF-β3, the ability to bind toactivin-A, the ability to bind to bone morphogenic protein(BMP)-2, theability to bind to BMP-7, the ability to bind to BMP-9, the ability tobind to BMP receptor II, the ability to bind TGF-β receptor I, theability to bind to TGF-β receptor II, the ability to reverse or inhibitangiogenesis, the ability to reduce or inhibit Smad 2/3-dependenttranscriptional activation, and the ability to inhibit endothelialnitric oxide synthase activation.

In various embodiments of the above aspect, the agent is a purifiedantibody or antibody binding fragment thereof that specifically binds toa soluble endoglin polypeptide, where the antibody or antibody-bindingfragment binds to an epitope that includes a sequence that is at least85% identical to SEQ ID NO: 2, or a fragment thereof. Desirably, theepitope fragment is at least 3 amino acids, 4 amino acids, 5 aminoacids, or 6 amino acids. In one embodiment, the antibody orantibody-binding fragment binds to an epitope that includes SEQ ID NO:2. In another embodiment, the antibody or antibody-binding fragmentcompetes with the antibody C-5144 for binding to soluble endoglin. Theantibody, or fragment thereof, can be a monoclonal antibody, a chimeric,a humanized, a human antibody, an antibody that lacks an Fe portion oris a F(ab′)₂, a Fab, or an Fv structure. In various embodiments, theantibody or antibody binding fragment is in a pharmaceuticallyacceptable carrier.

In another embodiment of the above aspect, the agent is an inhibitorynucleic acid molecule that includes at least one strand that iscomplementary to a nucleic acid sequence that encodes an amino acidsequence that is at least 85% identical to SEQ ID NO: 2, or a fragmentthereof, and wherein the inhibitory nucleic acid molecule reduces orinhibits the expression or a biological activity of a soluble endoglinpolypeptide. The inhibitory nucleic acid molecule desirably has at leastone strand that is complementary to a nucleic acid sequence that encodesthe amino acid sequence of SEQ ID NO: 2.

In one embodiment, the inhibitory nucleic acid molecule is adouble-stranded RNA, a small interfering RNA (siRNA) or a micro RNA. ThesiRNA can be between 17 to 25 nucleotides in length. In anotherembodiment, the isolated inhibitor y nucleic acid molecule issingle-stranded DNA or an antisense nucleobase oligomer.

In various embodiment of the above aspect, the a solubleendoglin-mediated disorder is selected from the group consisting ofpre-eclampsia, eclampsia, gestational hypertension, chronichypertension, HELLP syndrome, pregnancy with asmaller-for-gestational-age (SGA) infant, abruption pregnancy, pregnancywith intrauterine growth restriction, pulmonary hypertension, cancer(where soluble endoglin is elevated), and malaria. Additionalnon-limiting examples of soluble endoglin-mediated disorders includeAlzheimer's disease, cerebral arteriovenous malformations, leftventricular dysfunction, and patients with diabetes having retinopathyand/or a high probability of 10-year cardiovascular risk, as well aspatients with diabetes and hypertension who have three or more damagedtarget organs (e.g., heart, vessels, and kidney). Desirably, a solubleendoglin-mediated disorder is pre-eclampsia or eclampsia.

In various embodiments, the method results in at least a 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more reduction in one or more symptomsof a soluble endoglin-mediated disorder. In additional embodiments, themethod further includes administering one or more agents selected fromthe group of: an agent that increases endothelial nitric oxide synthaseexpression or biological activity, an agent that increases the levels ofprostacyclin or prostacyclin biological activity, an agent thatincreases TGF-β levels or biological activity, an agent that decreasessFlt-1 expression or biological activity, an agent that increasesvascular endothelial factor levels or biological activity, and an agentthat increases placenta growth factor levels or biological activity.

In another aspect, the invention features a method of treating ordecreasing the likelihood of developing a soluble endoglin-preventivedisorder in a subject, that includes the step of administering to thesubject an isolated soluble endoglin polypeptide having a solubleendoglin biological activity and comprising a sequence that is at least85% identical to VRWTVTC (SEQ ID NO: 2), or an isolated nucleic acidencoding the isolated soluble endoglin polypeptide.

In one embodiment, the sequence that is at least 85% identical to SEQ IDNO: 2 is located at the C-terminus of the soluble endoglin polypeptide.In another embodiment the soluble endoglin polypeptide includes thesequence of SEQ ID NO: 2, desirably at the C-terminus of the solubleendoglin.

In various embodiments, the isolated soluble endoglin polypeptideincludes or consists of an amino acid sequence that is at least 98%,99%, or 100% identical to SEQ ID NO: 3. The soluble endoglin polypeptidecan include fragments of soluble endoglin, for example, fragments thatinclude a total of at least 100, 200, 250, 300, 350, 400, or 444 aminoacids. The soluble endoglin polypeptide, or fragments thereof, canfurther include an additional amino acid sequence at the N- orC-terminus, which can be comprises at least 5, 10, 15, 20, 25 aminoacids or more.

In additional embodiments, the soluble endoglin polypeptide biologicalactivity is selected from the group consisting of: the ability to bindto TGF-β1, the ability to bind to TGF-β3, the ability to bind toactivin-A, the ability to bind to bone morphogenic protein(BMP)-2, theability to bind to BMP-7, the ability to bind to BMP-9, the ability tobind to BMP receptor II, the ability to bind TGF-β receptor I, theability to bind to TGF-β receptor II, the ability to reverse or inhibitangiogenesis, the ability to reduce or inhibit Smad 2/3-dependenttranscriptional activation, and the ability to inhibit endothelialnitric oxide synthase activation.

The isolated soluble endoglin polypeptide can further include one ormore modifications selected from the group consisting of: acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, S-nitrosylation, disulfide bond formation,demethylation, formation of cysteine, formation of sulfonic, sulfenic,or sulfinic acid, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids, ubiquitination,D-isomerization, derivitization of water soluble polymers, addition of adetectable label, and conjugation of another protein or therapeuticcompound. In one embodiment, the modification includes glycosylation ors-nitrosylation.

In various embodiments, the soluble endoglin-preventive disorderincludes a disorder characterized by factors (e.g. increased solubleendoglin or TGF-β1 or 3 levels or activity) or events (e.g. fibrosis,angiogenesis, immune activation) that contribute to the disorder and/orthat can be inhibited or modulated by soluble endoglin. Examples includefibrotic disorders of internal organs and the scarring of skin whereTGF-β is a significant contributor, disorders characterized by excessiveangiogenesis (e.g. hemangiomas, pulmonary capillary hemangiomatosis) orabnormal growth of blood vessel such as cancer (e.g., cancer of thebreast, prostate, colon, lung, head and neck, liver, kidney, renalsystem, and endometrium) and inflammatory and immune disorders.

In various embodiment of the above aspect, the solubleendoglin-preventive disorder is a disorder characterized by increasedTGF-β (e.g., TGF β1 or TGFβ3) or TGF β receptor levels. In anotherembodiment, the soluble endoglin-preventive disorder is cancer, and themethod further comprises administering to the subject one or more agentsselected from the group consisting of: a chemotherapeutic agent, anangiogenesis inhibitor, and an anti-proliferative compound.

Desirably, for any of the above therapeutic aspects, the subject is amammal, preferably a human.

In another aspect, the invention features a method of diagnosing asubject as having, or having the predisposition to develop, a solubleendoglin-mediated disorder or a soluble endoglin-preventive disorder,wherein the method includes the steps of:

(a) measuring the level of a soluble endoglin polypeptide having asoluble endoglin biological activity and including a sequence that is atleast 85% identical to VRWTVTC (SEQ ID NO: 2) in a sample from asubject;

(b) measuring the level of the soluble endoglin polypeptide in a controlsample; and

(c) comparing the level of the soluble endoglin polypeptide in thesubject to the level of the soluble endoglin polypeptide in the controlsample; wherein an increase in the level of the soluble endoglinpolypeptide in the subject as compared to the level of the solubleendoglin polypeptide in the control sample diagnoses the subject ashaving, or having a disposition for developing, a solubleendoglin-mediated disorder, or wherein a decrease in the level of thesoluble endoglin polypeptide in the subject as compared to the level ofthe soluble endoglin polypeptide in the control sample diagnosis thesubject as having, or having a disposition for developing, a solubleendoglin-preventive disorder.

In one embodiment, the sequence that is at least 85% identical to SEQ IDNO: 2 is located at the C-terminus of the soluble endoglin polypeptide.In another embodiment, the soluble endoglin polypeptide comprises anamino acid sequence that is at least 98% identical to SEQ ID NO: 3.

In various embodiments, the measuring in step (a) or step (b) isperformed using an antibody or antibody binding fragment thereof thatspecifically binds to a soluble endoglin polypeptide, wherein theantibody or antibody-binding fragment binds to an epitope comprising asequence that is at least 85% identical to SEQ ID NO: 2, or a fragmentthereof. Desirably, the fragment is at least 3 amino acids, 4 aminoacids, 5 amino acids, or 6 amino acids. In one embodiment, the antibodyor antibody-binding fragment binds to an epitope that includes SEQ IDNO: 2. In another embodiment, the antibody or antibody-binding fragmentcompetes with the antibody C-5144 for binding to soluble endoglin.

In one embodiment, the method includes the use of an immunoassay. Inanother embodiment, the control sample is a prior sample from thesubject. The sample from the subject or the control sample can be abodily fluid (e.g., urine, amniotic fluid, blood, serum, or plasma),cell, or a tissue sample.

In another aspect, the invention features a kit that includes: (a) anantibody or antibody binding fragment thereof that reduces the levels orbiological activity of a soluble endoglin polypeptide comprising asequence that is at least 85% identical to VRWTVTC (SEQ ID NO: 2),wherein the antibody or antibody binding fragment thereof specificallybinds to an epitope that includes a sequence that is at least 85%identical to SEQ ID NO: 2, or a fragment thereof; and (b) instructionsfor administering that antibody of (a) to a subject having a solubleendoglin-mediated disorder.

In one embodiment, the antibody or antibody-binding fragment binds to anepitope that includes the sequence of SEQ ID NO: 2. In anotherembodiment, the antibody is a monoclonal antibody or any antibodydescribed herein.

In another aspect, the invention features a kit that includes: (a) aninhibitory nucleic acid molecule that reduces the levels or biologicalactivity of a soluble endoglin polypeptide comprising a sequence that isat least 85% identical to VRWTVTC (SEQ ID NO: 2), wherein the inhibitorynucleic acid molecule includes at least one strand that is complementaryto a nucleic acid sequence that encodes an amino acid sequence that isat least 85% identical to VRWTVTC (SEQ ID NO: 2), and (b) instructionsfor administering the inhibitory nucleic acid molecule of (a) to asubject having a soluble endoglin-mediated disorder.

In another embodiment of the above aspect, the inhibitory nucleic acidmolecule includes at least one strand that is complementary to a nucleicacid sequence that encodes an amino acid sequence that is at least 85%identical to SEQ ID NO: 2, and wherein the inhibitory nucleic acidmolecule reduces or inhibits the expression or a biological activity ofa soluble endoglin polypeptide. The inhibitory nucleic acid moleculedesirably has at least one strand that is complementary to a nucleicacid sequence that encodes the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the inhibitory nucleic acid molecule is adouble-stranded RNA, a small interfering RNA (siRNA) or a micro RNA. ThesiRNA can be between 17 to 25 nucleotides in length. In anotherembodiment, the isolated inhibitor nucleic is single-stranded DNA or anantisense nucleobase oligomer.

In various embodiments of either of the above aspects, the kit furtherincludes one or more agents selected from the group of: an agent thatincreases endothelial nitric oxide synthase expression or biologicalactivity, an agent that increases the levels of prostacyclin orprostacyclin biological activity, an agent that increases TGF-βlevels orbiological activity, an agent that decreases sFlt-1 expression orbiological activity, an agent that increases vascular endothelial factorlevels or biological activity, and an agent that increases placentagrowth factor levels or biological activity.

In another aspect, the invention features a kit that includes (a) anisolated soluble endoglin polypeptide, wherein the soluble endoglinpolypeptide has a soluble endoglin biological activity and includes asequence that is at least 85% identical to VRWTVTC (SEQ ID NO: 2) or anucleic acid encoding the soluble endoglin polypeptide; and (b)instructions for administering the soluble endoglin polypeptide or thenucleic acid encoding the soluble endoglin polypeptide to a subjecthaving a soluble endoglin-preventive disorder. In one embodiment, thesequence that is at least 85% identical to SEQ ID NO: 2 is located atthe C-terminus of the soluble endoglin polypeptide. In anotherembodiment the soluble endoglin polypeptide includes the sequence of SEQID NO: 2, desirably at the C-terminus of the soluble endoglin.

In various embodiments, the isolated soluble endoglin polypeptideincludes or consists of an amino acid sequence that is at least 98%,99%, or 100% identical to SEQ ID NO: 3. The soluble endoglin polypeptidecan include fragments of soluble endoglin, for example, fragments thatinclude a total of at least 100, 200, 250, 300, 350, 400, or 444 aminoacids. The soluble endoglin polypeptide, or fragments thereof, canfurther include an additional amino acid sequence at the N- orC-terminus, which can be comprises at least 5, 10, 15, 20, 25 aminoacids or more.

In various embodiments, the kit further includes one or more agentsselected from the group of: a chemotherapeutic agent, an angiogenesisinhibitor, and an anti-proliferative compound.

By “binding” is meant a non-covalent or a covalent interaction,preferably non-covalent, that holds two molecules together. For example,two such molecules could be a ligand and its receptor, an enzyme and aninhibitor of that enzyme, an enzyme and its substrate, or an antibodyand an antigen. Non-covalent interactions include, but are not limitedto, hydrogen bonding, ionic interactions among charged groups, van derWaals interactions, and hydrophobic interactions among non-polar groups.One or more of these interactions can mediate the binding of twomolecules to each other. Binding may exhibit discriminatory propertiessuch as specificity or selectivity.

By “tumor” or “cancer” is meant both benign and malignant growths ofcancer. The cancer can be a non-solid tumor (e.g., a tumor that growswithin the blood stream) or a solid tumor, which refers to one thatgrows in an anatomical site outside the bloodstream (in contrast, forexample, to blood-borne tumors, such as lymphomas and leukemia) andrequires the formation of small blood vessels and capillaries to supplynutrients, etc., to the growing tumor mass. Examples of solid tumorsinclude tumors of the gastrointestinal tract, colon, breast, prostate,lung, kidney, liver, pancreas, ovary, head and neck, oral cavity,stomach, duodenum, small intestine, large intestine, anus, gall bladder,labium, nasopharynx, skin, uterus, male genital organ, urinary organ,bladder, skin, sarcomas, brain tumors, and bone tumors. The methods andcompositions of the invention may be used to treat or decrease thelikelihood of developing any type of cancer encompassed by thisdefinition of “cancer.”

By “chemotherapeutic agent” is meant a chemical that may be used todestroy a cancer cell, or to slow, arrest, or reverse the growth of acancer cell. Chemotherapeutic agents include, without limitation,anastrozole, asparaginase, azacitidine, bevacizumab, dicalutamide,bleomycin, bortezomib, busulfan carmustine (commonly referred to asBCNU), capecitabine, carboplatin, cetuximab, chlorambucil, cisplatin,cladribine (commonly referred to as 2-CdA), CPT11, cyclophosphamide,cytarabine (commonly referred to as Ara-C), dacarbazine, dasatinib,daunorubicin, dexamethasone, docetaxel, doxorubicin (commonly referredto as Adriamycin), epirubicin, erlotinib, exemestane, etoposide,fludarabine, 5-fluorouracil (commonly referred to as 5FU), gefitinib,gemcitabine, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,interferon-γ (native or recombinant), irinotecan, lapatinib, letrozole,levamisole, lomustine (commonly referred to as CCNU), mechlorethamine(commonly referred to as nitrogen mustard), melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel,pemetrexed, pentostatin, prednisone, procarbazine, rituximab, sorafenib,sunitinib, tamoxifen, taxol-related compounds, temozolomide,6-thioguanine, topotecan, trastuzumab, triptorelin, vinblastine,vincristine, and vinorelbine.

By “compound” is meant any small molecule chemical compound (peptidyl ornon-peptidyl), antibody, nucleic acid molecule, polypeptide, orfragments thereof. Compounds particularly useful for methods of treatingor decreasing the likelihood of developing a soluble endoglin-mediateddisorder preferably decrease the levels (protein or mRNA levels) or abiological activity of soluble endoglin polypeptide of the invention byat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,90%, 95%, or 99% or more. Assays for measuring the biological activityof soluble endoglin are described herein or are known in the art.Compounds particularly useful for methods of treating or decreasing thelikelihood of developing a soluble endoglin-preventive disorderpreferable increase the levels of soluble endoglin of the invention(protein or mRNA levels) or a biological activity of soluble endoglinpolypeptide of the invention by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99% or more.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495, 1975, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628, 1991 and Marks et al., J. Mol.Biol. 222:581-597, 1991, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855, 1984).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525, 1986; Riechmann et al., Nature 332:323-329, 1988; andPresta, Curr. Opin. Struct. Biol. 2:593-596, 1992.

The term “human antibody” or “fully human antibody” includes antibodieshaving variable and constant regions (if present) of human germlineimmunoglobulin sequences. Human antibodies of the invention can includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo) (see, Lonberg, N.et al. (1994) Nature 368(6474): 856-859); Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995)Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N.(1995) Ann. N.Y. Acad. Sci. 764:536-546). However, the term “humanantibody” does not include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences (i.e., humanizedantibodies).

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Ina preferred embodiment, the antibody mutant will have an amino acidsequence having at least 75% amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the species-dependent antibody, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e., same residue) orsimilar (i.e., amino acid residue from the same group based on commonside-chain properties, see below) with the species-dependent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

To increase the half-life of the antibodies or polypeptide containingthe amino acid sequences of this invention, one can attach a salvagereceptor binding epitope to the antibody (especially an antibodyfragment), as described, e.g., in U.S. Pat. No. 5,739,277. For example,a nucleic acid molecule encoding the salvage receptor binding epitopecan be linked in frame to a nucleic acid encoding a polypeptide sequenceof this invention so that the fusion protein expressed by the engineerednucleic acid molecule comprises the salvage receptor binding epitope anda polypeptide sequence of this invention. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule (e.g.,Ghetie et al., Ann. Rev. Immunol. 18:739-766, 2000, Table 1). Antibodieswith substitutions in an Fc region thereof and increased serumhalf-lives are also described in WO 00/42072; WO 02/060919; Shields, R.L., et al., J. Biol. Chem. 276(9):6591-6604, 2001; Hinton, P. R., J.Biol. Chem. 279(8):6213-6216, 2004). In another embodiment, the serumhalf-life can also be increased, for example, by attaching otherpolypeptide sequences. For example, antibodies of this invention orother polypeptide containing the amino acid sequences of this inventioncan be attached to serum albumin or a portion of serum albumin thatbinds to the FcRn receptor or a serum albumin binding peptide so thatserum albumin binds to the antibody or polypeptide, e.g., suchpolypeptide sequences are disclosed in WO 01/45746. In one preferredembodiment, the serum albumin peptide to be attached comprises an aminoacid sequence of DICLPRWGCLW (SEQ ID NO: 5). In another embodiment, thehalf-life of a Fab according to this invention is increased by thesemethods. See also, Dennis, M. S., et al., J. Biol. Chem.277(38):35035-35043, 2002 for serum albumin binding peptide sequences.

By “double-stranded RNA (dsRNA)” is meant a ribonucleic acid moleculecomprised of both a sense and an anti-sense strand. dsRNAs are typicallyused to mediate RNA interference.

By “endoglin” or “Eng,” also known as CD105, is meant a mammalian growthfactor that has endoglin biological activity (see, Fonsatti et al.,Oncogene 22:6557-6563, 2003; Fonsatti et al., Curr. Cancer Drug Targets3:427-432, 2003; and Cheifetz et al., J. Biol. Chem. 267:19027-19030(1992)) and is homologous to the protein defined by any of the followingGenBank Accession Numbers: AAH29080 and NP_(—)031958 (mouse); AAS67893(rat); NP_(—)000109, P1781, VSP_(—)004233, and CAA80673 (pig); andCAA50891 and AAC63386 (human); or the sequences described in U.S. Pat.No. 6,562,957. Endoglin is a homodimeric cell membrane glycoproteinwhich is expressed at high levels in proliferating vascular endothelialcells and in the syncytiotrophoblasts from placentas. There are twodistinct isoforms of endoglin, L and S, which differ in theircytoplasmic tails by 47 amino acids. Both isoforms are included in theterm endoglin as used herein. Endoglin binds to TGF-β family membersand, in the presence of TGF-β, endoglin can associate with the TGF-βsignaling receptors RI and RII or BMP receptor II, and potentiate theresponse to the growth factors. Endoglin biological activities include:binding to substrates such as TGF-β family members, such as activin-A,BMP-2, BMP-7, BMP-9, TGF-β1, and TGF-β3; induction of angiogenesis;regulation of cell proliferation, attachment, migration, and invasion;and activation of endothelial cells. Assays for endoglin biologicalactivities are known in the art and are described in WO 07/143,023 andWO 08/030,283 (each incorporated by reference), and include withoutlimitation ligand binding assays or Scatchard plot analysis; BrdUlabeling, cell counting experiments, or quantitative assays for DNAsynthesis such as ³H-thymidine incorporation used to measure cellproliferation; and angiogenesis assays such as those described inMcCarty et al., Intl. Oncol. 21:5-10, 2002; Akhtar et al. Clin. Chem.49:32-40, 2003; and Yamashita et al, Biol. Chem. 269:1995-2001, 1994.

By “soluble endoglin polypeptide” or “sEng” is meant a circulating,non-membrane bound form of endoglin which includes at least a part ofthe extracellular portion of the endoglin protein, contains a sequencethat is substantially identical (e.g., at least 70%, 71%, 72%, 73%, 74%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to the sequence of VRWTVTC (SEQ ID NO: 2), and has a solubleendoglin biological activity. In one embodiment, the soluble endoglinpolypeptide contains the sequence of SEQ ID NO: 1. Desirable forms ofsoluble endoglin polypeptides contain the sequence of SEQ ID NO: 1 orSEQ ID NO: 2 and have a soluble endoglin biological activity. The formsof soluble endoglin polypeptide encompassed by this term may be producedfrom the alternate splicing of an endoglin precursor mRNA and may bemonomeric. For example, one non-limiting example of a soluble endoglinpolypeptide is depicted in FIG. 1 (cDNA and polypeptide sequencescorresponding to SEQ ID NOS: 4 and 3, respectively). Non-limitingexamples of soluble endoglin polypeptides of the invention contain asequence that is substantially identical (e.g., at least 70%, 75%, 85%,90%, 95%, or even 100% identical) to SEQ ID NO: 1 or SEQ ID NO: 2 (e.g.,may contain one or two conservative mutations in the sequence of SEQ IDNO: 1 or SEQ ID NO: 2), have a soluble endoglin biological activity, andmay also contain a sequence that is at least 85% identical (e.g., atleast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the sequenceof SEQ ID NO: 3 (upon comparing an equal number of amino acids in thesequence of the soluble endoglin polypeptide to an equal number of aminoacids in the sequence of SEQ ID NO: 3). Preferred soluble endoglinpolypeptides contain a sequence at least 85% identical (e.g., at least90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 1 or SEQID NO: 2 at the C-terminus.

Soluble endoglin polypeptides include a sequence that is substantiallyidentical to SEQ ID NO: 2, have a soluble endoglin biological activity,and may have a total length of at least 10 amino acids (e.g., at least20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, or 444 aminoacids). Soluble endoglin polypeptides of the invention may contain atleast 444 amino acids. The soluble endoglin polypeptides of theinvention may also contain additional amino acids sequences at the N-and/or C-terminus of the polypeptide.

The soluble endoglin polypeptides of the invention have a solubleendoglin polypeptide biological activity such as, but limited to,binding to substrates such as TGF-β family members (e.g., TGF-(31,TGF-β3, activin-A, BMP-2, BMP-7, and BMP-9), TGF-β receptors (e.g.,TGF-β receptor I and TGF-β receptor II), or BMP receptor II reducing orinhibiting the activation of Smad 2/3 or Smad 2/3-dependenttranscriptional activation, inhibiting the biological activity of TGF-βfamily members, reversing or inhibiting angiogenesis induced by TGF-β byat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%,80%, 90%, or more, or inhibiting endothelial nitric oxide synthaseactivation. Examples of assays for measuring these activities are knownin the art and described in U.S. Patent Application Publication Nos.20060067937, 20050267021, and 20070104707 and PCT Publication Nos. WO06/034507, WO 07/143,023, and WO 08/030,283 (each incorporated herein byreference). Soluble endoglin polypeptides may be isolated from a varietyof sources, such as from mammalian tissue or cells (e.g., placentaltissue or cells), or prepared by recombinant or synthetic methods. Theterm soluble endoglin polypeptide can also encompass modifications tothe polypeptide or fragments of the soluble endoglin polypeptide, andderivatives and analogues thereof, examples of which are describedbelow.

By “soluble endoglin nucleic acid” is meant a nucleic acid that encodesany of the soluble endoglin polypeptides of the invention. Anon-limiting example of a nucleic acid that encodes a soluble endoglinpolypeptide is shown in FIG. 1 (SEQ ID NO: 4). Desirably, the solubleendoglin nucleic acid encodes a soluble endoglin polypeptide containinga sequence that is at least 85% identical to SEQ ID NO: 2 that has asoluble endoglin biological activity. Non-limiting examples of solubleendoglin nucleic acids contain a sequence that is substantiallyidentical (at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to the nucleic acid sequence set forth in FIG. 1 (SEQ ID NO:4) and contain a sequence that encodes a polypeptide sequence that has asoluble endoglin biological activity and contain a sequence that is atleast 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%identical) to SEQ ID NO: 1 or SEQ ID NO: 2.

By “soluble endoglin-mediated disorder” is a disorder where an increasein soluble endoglin levels or an increase in soluble endoglin biologicalactivity has been detected or implicated in the pathogenesis ordevelopment of the disorder. For example, a subject having an solubleendoglin-mediated disorder may have an increase (e.g., at least a 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or even 100% increase) in the expression level (e.g., polypeptide ormRNA levels) of soluble endoglin or may have an increase (e.g., at least5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%increase) in a biological activity of a soluble endoglin polypeptide asdescribed herein. Non-limiting examples of soluble endoglin-mediateddisorders include pregnancy-related hypertensive disorders (e.g.,pre-eclampsia, eclampsia, gestational hypertension, chronichypertension, HELLP syndrome, and pregnancy with asmaller-for-gestational-age (SGA) infant), pulmonary hypertension,cancer (where the soluble endoglin levels are elevated), and malaria.Additional examples include Alzheimer's disease, cerebral arteriovenousmalformations, and left ventricular dysfunction. In addition, patientswith diabetes having retinopathy and/or a high probability of 10-yearcardiovascular risk and patients with diabetes and hypertension who havethree or more damaged target organs (e.g., heart, vessels, and kidney)are also included in some embodiments.

By “soluble endoglin-preventive disorder” is meant a disorder where adecrease in the soluble endoglin levels (protein or mRNA) or the solubleendoglin biological activity has been detected or implicated in thepathogenesis of the disorder or where specific factors, such as TGF-βfamily proteins or TGFβ family receptors or events (e.g. fibrosis,angiogenesis, immune activation) that can be inhibited or modulated bysoluble endoglin, contribute to the disorder. Non-limiting examplesinclude fibrotic disorders of internal organs and the scarring of skinwhere TGF-β is a significant contributor, disorders characterized byexcessive angiogenesis (e.g. hemangiomas, pulmonary capillaryhemagiomatosis) or abnormal growth of blood vessel such as cancer (e.g.,cancer of the breast, prostate, colon, lung, head and neck, liver,kidney, renal system, and endometrium) and inflammatory and immunedisorders (e.g. excessive TGF-β may be related to autoimmune diseases).In one non-limiting example, a subject having a solubleendoglin-preventive disorder may have a decrease (e.g., at least 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100% decrease) in the expression level (e.g., polypeptide or mRNAlevels) of soluble endoglin or may have a decrease (e.g., at least 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or even 100% decrease) in a biological activity of a soluble endoglinpolypeptide as compared to a normal reference as described herein.

By “epitope” is meant a sequence of amino acids which, either as aresult of linear structure or three dimensional conformation, forms thebinding site for an antibody.

By “expression” is meant the detection of a gene or polypeptide bystandard art known methods. For example, polypeptide expression is oftendetected by Western blotting, DNA expression is often detected bySouthern blotting or polymerase chain reaction (PCR), and RNA expressionis often detected by northern blotting, PCR, or RNAse protection assays.Methods to measure protein expression levels generally include, but arenot limited to: Western blot, immunoblot, enzyme-linked immunosorbantassay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surfaceplasmon resonance, chemiluminescence, fluorescent polarization,phosphorescence, immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,microcytometry, microarray, microscopy, fluorescence activated cellsorting (FACS), and flow cytometry, as well as assays based on aproperty of the protein including but not limited to enzymatic activityor interaction with other protein partners. Exemplary assays aredescribed in detail in U.S. Patent Application Publication No.2006/0067937 and PCT Publication Nos. WO 06/034507, WO 07/030,283, andWO 08/030,283. Any compound that decreases the presently describedsoluble endoglin polypeptide or nucleic acid expression levels by atleast 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or more) is desirable for methods of treatingor decreasing the likelihood of developing a soluble endoglin-mediateddisorder. Any compound that increases the presently described solubleendoglin polypeptide or nucleic acid expression levels by at least 5%(e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or more) is desirable for methods of treating ordecreasing the likelihood of developing a soluble endoglin-preventivedisorder.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%of the entire length of the soluble endoglin nucleic acid molecule(e.g., a sequence that is at least 95% identical to SEQ ID NO: 4) orpolypeptide of the invention (e.g., a sequence that is at least 95%identical to SEQ ID NO: 3). A fragment of a soluble endoglin nucleicacid of the invention may contain at least 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1332, or more nucleotides or at least 6, 7,8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, or 440 aminoacids or more. The fragments of a soluble endoglin polypeptide have asoluble endoglin polypeptide activity and contain a sequence that issubstantially identical (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to SEQ ID NO: 1 or SEQ ID NO: 2. The fragments of a nucleicacid encoding a soluble endoglin polypeptide desirably contain a nucleicacid sequence that encodes a polypeptide sequence that has a solubleendoglin biological activity and contains a sequence that issubstantially identical (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to SEQ ID NO: 1 or SEQ ID NO: 2. Exemplary fragments ofsoluble endoglin polypeptide have a soluble endoglin biological activityand include a contiguous amino acid sequence from 1 to 444 amino acids(e.g., SEQ ID NO: 3) (including the peptide leader sequence) or acontiguous amino acid sequence from 1 to 418 amino acids (e.g.,fragments excluding amino acids 1 to 25 of SEQ ID NO: 3, as shown inFIGS. 3A and 3B). Additional exemplary fragments have a soluble endoglinbiological activity and contain a sequence identical to SEQ ID NO: 1 orSEQ ID NO: 2 at the C-terminus.

By “gestational hypertension” is meant the development of high bloodpressure without proteinuria after 20 weeks of pregnancy.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

By “metastatic disease,” “metastases,” and “metastatic lesion” are meanta group of cells which have migrated to a site distant relative to theprimary tumor. “Non-metastatic” refers to tumor cells, e.g., humancancer cells, that are unable to establish secondary tumor lesionsdistant to the primary tumor. Although not often the case, metastaticdisease can occur when no primary tumor has been detected. The cells ina metastatic tumor resemble those in the primary tumor. Metastasis ormetastatic disease can be diagnosed in a variety of ways that are knownin the art.

By “nitric oxide synthase” or “NOS” is meant an enzyme that catalyzesthe formation of nitric oxide (NO) from oxygen and arginine. NOS is acomplex enzyme containing several cofactors, a heme group which is partof the catalytic site, an N-terminal oxygenase domain, which belongs tothe class of heme-thiolate proteins, and a C-terminal reductase domainwhich is homologous to NADPH:P450 reductase. NOS produces NO bycatalyzing a five-electron oxidation of a guanidino nitrogen ofL-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via twosuccessive monooxygenation reactions producing N-hydroxy-L-arginine asan intermediate. The interdomain linker between the oxygenase andreductase domains contains a calmodulin-binding sequence. NO functionsat low concentrations as a signal in many diverse physiologicalprocesses such as blood pressure control, neurotransmission, learningand memory, and at high concentrations as a defensive cytotoxin.

In mammals, three distinct genes encode NOS isozymes: neuronal NOS (nNOSor NOS-1), cytokine-inducible NOS (iNOS or NOS-2), and endothelial NOS(eNOS or NOS-3). eNOS is membrane associated and its localization toendothelial membranes is mediated by cotranslational N-terminalmyristoylation and post-translational palmitoylation. In preferredembodiments of the invention, the NOS is eNOS.

By “operably-linked” is meant that a gene and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s).

By “pharmaceutically acceptable carrier” is meant a carrier that isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier substance isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to one skilled in the art and described,for example, in Remington's Pharmaceutical Sciences, (20^(th) edition),ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.One or more of the soluble endoglin polypeptides or nucleic acids of theinvention may be administered in a pharmaceutically acceptable carrierto a subject (e.g., a human).

By “pregnancy-related hypertensive disorder” is meant any condition ordisease during pregnancy that is associated with or characterized by anincrease in blood pressure. Included among these conditions arepre-eclampsia (including premature pre-eclampsia, severe pre-eclampsia),eclampsia, gestational hypertension, HELLP syndrome, (hemolysis,elevated liver enzymes, low platelets), abruption placenta, chronichypertension, pregnancy with intrauterine growth restriction, andpregnancy with a small for gestational age (SGA) infant. It should benoted that although pregnancy with a SGA infant is not often associatedwith hypertension, it is included in this definition.

By “pre-eclampsia” is meant the multi-system disorder that ischaracterized by hypertension with proteinuria or edema, or both,glomerular dysfunction, brain edema, liver edema, or coagulationabnormalities due to pregnancy or the influence of a recent pregnancy.All forms of pre-eclampsia, such as premature, mild, moderate, andsevere pre-eclampsia are included in this definition. Pre-eclampsiagenerally occurs after the 20^(th) week of gestation. Pre-eclampsia isgenerally defined as some combination of the following symptoms: (1) asystolic blood pressure (BP)>140 mmHg and a diastolic BP>90 mmHg after20 weeks gestation (generally measured on two occasions, 4-168 hoursapart), (2) new onset proteinuria (1+by dipstik on urinalysis, >300 mgof protein in a 24-hour urine collection, or a single random urinesample having a protein/creatinine ratio>0.3), and (3) resolution ofhypertension and proteinuria by 12 weeks postpartum. Severepre-eclampsia is generally defined as (1) a diastolic BP>110 mmHg(generally measured on two occasions, 4-168 hours apart) or (2)proteinuria characterized by a measurement of 3.5 grams or more proteinin a 24-hour urine collection or two random urine specimens with atleast 3+ protein by dipstick. In pre-eclampsia, hypertension andproteinuria generally occur within seven days of each other. In severepre-eclampsia, severe hypertension, severe proteinuria and HELLPsyndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsiacan occur simultaneously or only one symptom at a time. HELLP syndromeis characterized by evidence of thrombocytopenia (<100,000 cells/μl),increased LDH (>600 IU/L), and increased AST (>70 IU/L). Occasionally,severe pre-eclampsia can lead to the development of seizures. Thissevere form of the syndrome is referred to as “eclampsia.” Eclampsia canalso include dysfunction or damage to several organs or tissues such asthe liver (e.g., hepatocellular damage and periportal necrosis) and thecentral nervous system (e.g., cerebral edema and cerebral hemorrhage).The etiology of the seizures is thought to be secondary to thedevelopment of cerebral edema and focal spasm of small blood vessels inthe kidney.

By “premature pre-eclampsia” is meant pre-eclampsia with onset ofsymptoms <37 weeks or <34 weeks.

By “prostacyclin” or “PGI₂” is meant a member of the family of lipidmolecules known as eicosanoids. It is produced in endothelial cells fromprostaglandin H2 (PGH2) by the action of the enzyme prostacyclinsynthase and is mainly synthesized by the vascular endothelium and bysmooth muscle. PGI₂ biological activity includes inhibition of plateletaggregation, relaxation of smooth muscle, reduction of systemic andpulmonary vascular resistance by direct vasodilation, and natriuresis inthe kidney.

By “purified” or “isolated” is meant separated from other componentsthat naturally accompany it. Typically, a compound (e.g., nucleic acid,polypeptide, or small molecule) is isolated when it is at least 50%, byweight, free from proteins, flanking nucleic acids, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably, the factor is at least 75%, more preferably, atleast 80%, 85%, or 90%, and most preferably, at least 95% or 99%, byweight, isolated. An isolated factor may be obtained by chemicalsynthesis, separation of the factor from natural sources, or productionof the factor in a recombinant host cell that does not naturally producethe factor. Proteins and small molecules may be purified by one skilledin the art using standard techniques such as those described by Ausubelet al. (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, 2000). The factor is preferably at least 2, 3, 4, 5, or 10 timesas pure or isolated as the starting material, as measured usingpolyacrylamide gel electrophoresis, column chromatography, opticaldensity, HPLC analysis, or Western blot analysis (Ausubel et al.,supra). Preferred methods of purification include immunoprecipitation,column chromatography (such as immunoaffinity chromatography), magneticbead immunoaffinity purification, and panning with a plate-boundantibody.

By “reduce or inhibit” is meant the ability to cause an overall decreasepreferably of at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50% or more, and more preferably an overall decrease of atleast 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. For example, in someembodiments of the invention, reduce or inhibit can refer to the levels(polypeptide or mRNA levels) or a biological activity of a solubleendoglin polypeptide of the invention, symptoms of the disorder beingtreated, the presence or size of metastases, the size of the primarytumor, or a biological activity of a TGF-β family member.

By “control” or “reference” is meant any sample, standard, or level thatis used for comparison purposes. For diagnostic or therapeuticmonitoring purposes, a control sample may be a prior sample taken fromthe same subject (e.g., a sample harvested at a prior time point orprior to the onset of symptoms). Non-limiting examples of controlsamples include: a sample from a pregnant subject or group of subjectsnot having a pregnancy related hypertensive disorder, such aspre-eclampsia or eclampsia; a subject or group of subjects that ispregnant but the sample was taken early in pregnancy (e.g., in the firstor second trimester or before the detection of a pregnancy relatedhypertensive disorder, such as pre-eclampsia or eclampsia); a subject orgroup of subjects that is pregnant and has no history of apregnancy-related hypertensive disorder, such as pre-eclampsia oreclampsia; a subject or group of subjects that is not pregnant; a samplefrom a subject or group of subjects not having a solubleendoglin-preventive disorder (e.g., cancer or a cardiovasculardisorder); a sample of a purified reference polypeptide at a knownnormal concentration (i.e., not indicative of a solubleendoglin-mediated disorder, such as pre-eclampsia or eclampsia, or notindicative of a soluble endoglin-preventive disorder). Additionalexamples of control samples can be prepared from a subject or group ofsubjects prior to developing or diagnosis with a solubleendoglin-mediated disorder or a soluble endoglin-preventive disorder(e.g., prior to the onset of one or more symptoms of a solubleendoglin-mediated disorder or a soluble endoglin-preventive disorder).By “control standard or level” is meant a value or number derived from acontrol sample. For example, a control standard or level can be a valueor number derived from a normal subject or group of subjects that ismatched to the sample subject, for example, by at least one of thefollowing criteria: age, sex, weight, gestational age of the fetus,maternal age, maternal blood pressure prior to pregnancy, maternal bloodpressure during pregnancy, BMI of the mother, weight of the fetus, priordiagnosis of pre-eclampsia or eclampsia, and a family history ofpre-eclampsia or eclampsia. A “positive control” sample, standard orvalue is a sample or value or number derived from a subject or group ofsubjects that is known to have a soluble endoglin-mediated disorder or asoluble endoglin-preventive disorder. For example, a positive controlsample may be from a subject or group of subjects having apregnancy-related hypertensive disorder (e.g., pre-eclampsia oreclampsia), that is matched to the sample subject by at least one of thefollowing criteria: gestational age of the fetus, maternal age, maternalblood pressure prior to pregnancy, maternal blood pressure duringpregnancy, BMI of the mother, weight of the fetus, prior diagnosis of apregnancy-related hypertensive disorder, and a family history of apregnancy related hypertensive disorder. Additional positive controlsmay be from a subject of group of subjects having the same solubleendoglin-mediated disorder or soluble endoglin-preventive disorder thatis matched for one or more criteria, such as sex, age, and weight.

By “sample” is meant a tissue biopsy, cell (e.g., endothelial cell ortumor cell), bodily fluid (e.g., blood, serum, plasma, urine, saliva,amniotic fluid, or cerebrospinal fluid) or other specimen obtained froma subject. Desirably, the biological sample includes soluble endoglinnucleic acids or polypeptides of the invention, or both.

By “small RNA” is meant an isolated RNA molecule, either single-strandedor double stranded that is at least 15 nucleotides, preferably, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35nucleotides in length and even up to 50 or 100 nucleotides in length(inclusive of all integers in between). Preferably, the small RNA iscapable of mediating RNAi. As used herein the phrase “mediates RNAi”refers to (indicates) the ability to distinguish which RNAs are to bedegraded by the RNAi machinery or process. Included within the termsmall RNA are “small interfering RNAs” and “microRNA.” In general,microRNAs (miRNAs) are small (e.g., 17-26 nucleotides), single-strandednoncoding RNAs that are processed from approximately 70 nucleotidehairpin precursor RNAs by Dicer. Small interfering RNAs (siRNAs) are ofsimilar size and are also non-coding, however, siRNAs are processed fromlong dsRNAs and are usually double-stranded (e.g., endogenous siRNAs).siRNAs can also include short hairpin RNAs in which both strands of ansiRNA duplex are included within a single RNA molecule. Small RNAs canbe used to describe both types of RNA. These terms includedouble-stranded RNA, single-stranded RNA, isolated RNA (partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA), as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution, and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the small RNA orinternally (at one or more nucleotides of the RNA). Nucleotides in theRNA molecules of the present invention can also comprise non-standardnucleotides, including non-naturally occurring nucleotides ordeoxyribonucleotides. Small RNAs of the present invention need only besufficiently similar to natural RNA that it has the ability to mediateRNAi.

By “specifically binds” is meant a compound or antibody which recognizesand binds a soluble endoglin polypeptide or nucleic acid of theinvention, but that does not substantially recognize and bind othermolecules in present in a sample (e.g., a biological sample whichnaturally includes a soluble endoglin polypeptide or nucleic acid of theinvention). In one example, an antibody that specifically binds asoluble endoglin polypeptide of the invention does not bindmembrane-bound endoglin or other forms of soluble endoglin. Desirably,an antibody or antibody-binding fragment thereof specifically binds toan epitope containing a sequence that is at least 70% identical (e.g.,at least 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical) to the sequence of SEQ IDNO: 1 or SEQ ID NO: 2, or a fragment thereof (e.g., 3 amino acids, 4amino acids, 5 amino acids, or 6 amino acids).

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a cow, a horse, a sheep, a pig, a goat, adog, or a cat. Included in this definition are pregnant, post-partum,and non-pregnant mammals.

By “substantially identical” is meant a nucleic acid or amino acidsequence that, when optimally aligned, for example using the methodsdescribed below, share at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% sequenceidentity with a second nucleic acid or amino acid sequence, e.g., asoluble endoglin sequence such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO: 4. “Substantial identity” may be used to refer tovarious types and lengths of sequence, such as a full-length sequence,epitopes or immunogenic peptides, functional domains, coding and/orregulatory sequences, exons, introns, promoters, and genomic sequences.Percent identity between two polypeptides or nucleic acid sequences isdetermined in various ways that are within the skill in the art, forinstance, using publicly available computer software such as SmithWaterman Alignment (Smith, T. F. and M. S. Waterman, J. Mod. Biol.147:195-7, 1981); “BestFit” (Smith and Waterman, Advances in AppliedMathematics, 482-489, 1981) as incorporated into GeneMatcher Plus™;BLAST program (Basic Local Alignment Search Tool), BLAST-2, BLAST-P,BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign(DNASTAR) software. In addition, those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the length of the sequencesbeing compared. In general, for proteins, the length of comparisonsequences will be at least 6 amino acids, preferably at least 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, or444 amino acids, or more. For nucleic acids, the length of comparisonsequences will generally be at least 20, 25, 30, 50, 100, 125, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, or 1330 nucleotides, ormore. It is understood that for the purposes of determining sequenceidentity when comparing a DNA sequence to an RNA sequence, a thyminenucleotide is equivalent to a uracil nucleotide. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

By “transforming growth factor β (TGF-β)” is meant a mammalian growthfactor that has TGF-β biological activity and is a member of a family ofstructurally related paracrine polypeptides found ubiquitously invertebrates, and prototypic of a large family of metazoan growth,differentiation, and morphogenesis factors (see, for review, Massaque etal. Ann. Rev. Cell. Biol. 6:597-641, 1990; Massaque et al. Trends Cell.Biol. 4:172-178, 1994; Kingsley Gene Dev. 8:133-146, 1994; and Sporn etal. J. Cell. Biol. 119:1017-1021, 1992. As described in Kingsley, supra,the TGF-β superfamily has at least 25 members, and can be grouped intodistinct sub-families with highly related sequences. The most obvioussub-families include the following: the TGF-β subfamily, which comprisesat least four genes that are much more similar to TGF-β1 than to othermembers of the TGF-β superfamily; the bone morphogenetic proteins; theactivin sub-family, comprising homo- or hetero-dimers or two subunits,inhibinβ-A and inhibinβ-B. The decapentaplegic subfamily, which includesthe mammalian factors BMP2 and BMP4, can induce the formation of ectopicbone and cartilage when implanted under the skin or into muscles. The60A subfamily includes a number of mammalian homologs withosteoinductive activity, including BMP5-8. Other members of the TGF-βsuperfamily include the gross differentiation factor 1 (GDF-1),GDF-3/VGR-2, dorsalin, nodal, mullerian-inhibiting substance (MIS), andglial-derived neurotrophic growth factor (GDNF). It is noted that theDPP and 60A subfamilies are related more closely to one another than toother members of the TGF-β superfamily, and have often been groupedtogether as part of a larger collection of molecules called DVR (dpp-and vgl-related). Unless evidenced from the context in which it is used,the term TGF-β as used throughout this specification will be understoodto generally refer to members of the TGF-β superfamily as appropriate(Massague et al., Anna. Rev. Biochem. 67:753-91, 1998; Josso et al.,Curr. Op. Gen. Dev., 7:371-377, 1997). TGF-β functions to regulategrowth, differentiation, motility, tissue remodeling, neurogenesis,wound repair, apoptosis, and angiogenesis in many cell types. TGF-β alsoinhibits cell proliferation in many cell types and can stimulate thesynthesis of matrix proteins.

By “therapeutic amount” is meant an amount that when administered,either by direct administration or by an ex vivo approach, to a patientsuffering from a soluble endoglin-preventive disorder or a solubleendoglin-mediated disorder is sufficient to cause a qualitative orquantitative reduction (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or even 100% reduction) in oneor more (e.g., 2, 3, 4, 5, or 6) of the symptoms of the solubleendoglin-preventive disorder or the soluble endoglin-mediated disorder(described below). A therapeutic amount may also mean an amount thatwhen administered results in a decrease (e.g., at least a 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or even 100%reduction) in the likelihood of developing a soluble endoglin-preventivedisorder or a soluble endoglin-mediated disorder.

By “treating” or “ameliorating” is meant treating or ameliorating acondition or symptom(s) of the condition (e.g., the symptoms of solubleendoglin-preventive disorders or the symptoms of solubleendoglin-mediated disorders described herein). To “treat disease” or usefor “therapeutic treatment” refers to administering the treatment to asubject already suffering from a disease to improve the subject'scondition. Preferably, the subject is diagnosed with or identified ashaving a predisposition for developing a soluble endoglin-mediateddisorder or a soluble endoglin-preventive disorder. As compared with anequivalent untreated control, such amelioration or degree of treatmentis at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,or 100%, as measured by any standard technique.

By “vector” is meant a DNA molecule, usually derived from a plasmid orbacteriophage, into which fragments of DNA may be inserted or cloned. Arecombinant vector will contain one or more unique restriction sites,and may be capable of autonomous replication in a defined host orvehicle organism such that the cloned sequence is reproducible. A vectorcontains a promoter operably-linked to a gene or coding region suchthat, upon transfection into a recipient cell, an RNA or an encodedprotein or is expressed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleic acid (SEQ ID NO: 4) and amino acid sequenceof full length human soluble endoglin (HsEng) (SEQ ID NO: 3).

FIG. 2A is a schematic diagram that shows the human endoglin mRNAcomprised of 14 exons and the alternatively spliced human Eng-mRNAtranscript, which contains a unique 3′ sequence and UTR. The HsEng mRNAtranscript is generated by exon extension whereby exon 9b is “extended”by 372 bases into intron 10. cDNA sequencing revealed that this novel1683-base Eng mRNA transcript is comprised of the same translation startsite as the full-length form of Eng but harbors a stop codon at position1333 and 351 bp of untranslated region (UTR). FIG. 2B is a graph showingthe expression of the HsEng transcript in human tissues relative to 18SRNA levels using Taqman primer/probe sets. HsEng transcript is enrichedin human lung, placenta, uterus and testes.

FIGS. 3A and 3B are schematic diagrams showing the organization of theorphan and zona pellucida (ZP) domains, as well as thetransmembrane/cytoplasmic region of endoglin expressed on the plasmamembrane of cells. The signal peptide consisting of the first 25 aminoacids is cleaved upon expression of the protein on surface of cells. Aputative proteolytic cleavage site has been suggested at Arg437 withinthe ZP domain. HsEng protein consists of 444-amino acid and harbors aunique peptide, -VRWTVTC-, as a result of alternative pre-mRNA splicing.The same signal peptide (Met1-Ala25) is cleaved and is necessary for itsexpression. FIG. 3C is a representative IP-western blot showing theexpression of soluble endoglin protein with an apparent molecular weightof ˜64 kD under both non-reducing and reducing conditions in thecultured media taken after overexpression of soluble endoglin cDNA inbovine aortic endothelial cells. While a relatively small portion ofsEng was also detected as a ˜125 kD dimer, it exists predominantly as amonomer. FIG. 3D is a representative IP-western blot showing thedetection of soluble endoglin (HsEng) using an antibody (C-5144) to thenovel carboxy terminal sequence unique to soluble endoglin. An increasein soluble endoglin was specifically detected in the sera of patientswith pre-eclampsia (PE) vs. normal controls (C).

FIG. 4 shows a representative IP-Western blot showing significantlyelevated HsEng levels in sera of patients with PAH. HsEng detected inthese serum samples had the same apparent molecular weight (˜64 kD) asthe positive control, consisting of the conditioned medium of HEK293Tcells transfected with HsEng cDNA.

FIGS. 5A-5D are graphs showing the change in peak RVSP (5A), RVhypertrophy (5B), RV delta pressure/delta time (dp/dt) value (5C), andRV contractility index (5D) in mice inoculated with Ad-Empty (control)or Ad-HsEng and then exposed to hypoxic conditions for either 3 weeks or6 weeks.

FIG. 6 is a representative IP-Western blot showing that cellstransfected with pc-HsEng (expressing the 444 amino acid form of solubleendoglin) produced a stable and glycosylated 64 kD protein in thecondition medium. A prominent band at ˜50 kD was present in thecorresponding cell extracts and likely represents the unglycosylatedHsEng protein (expected: 49 kD). Cells transfected with HsEng-437 didnot give rise to a 64 kD protein in the condition media, while a faintband was observed in the corresponding cell extracts at ˜50 kD(unglycosylated protein).

FIG. 7 shows a representative western blot and graph ofimmunoprecipitates from untreated samples and those treated with GSNOand subjected to biotin-switch. The expression of HsEng in HEK293T cellsthat do not normally express eNOS (enzyme that produces NO.), resultedin equal amounts of dimeric and monomeric HsEng (left panel). Ex vivotreatment of HsEng produced in HEK293T cells with the NO donor, GSNO,resulted in the S-nitrosylation of HsEng. S-nitrosylation of HsEngreduced the dimer/monomer ratio of this protein and may potentiallyimpact its function.

FIGS. 8A-8C show a schematic for the generation of the tetracyclineinducible transgenic mouse. FIG. 8D shows the results of genotyping ofthese mice using PCR primers. Shown is a 331 bp amplicon encompassingthe distal end of pTRE-tight promoter and proximal end of the HsEngsequence.

FIGS. 9A-9B show representative graphs and a blot showing HsEng mRNAlevels (9A) and HsEng protein levels (9B) in the condition media aftertreatment of HMVEC-L with TNF-α.

DETAILED DESCRIPTION

We have discovered a form of soluble endoglin produced by alternativesplicing of the endoglin precursor mRNA. We have isolated thispreviously unrecognized mRNA transcript of endoglin from human tissueand discovered that it encodes a stable and soluble (secreted) protein.This soluble form of endoglin exists predominantly as a monomer and isable to modulate TGF-13/BMP signaling. The soluble endoglin describedherein includes a unique C-terminal amino acid sequence of amino acids437-444 of RVRWTVTC (SEQ ID NO: 1) or amino acids 438-444 of VRWTVTC(SEQ ID NO: 2) and has a soluble endoglin biological activity. Theunique C-terminal amino acid sequences and the complete amino acid andnucleic acid sequences of the alternatively spliced mRNA transcript ofsoluble endoglin are shown in FIGS. 2A, 3B, and 1, respectively.

Soluble Endoglin Polypeptides

The invention features isolated forms of soluble endoglin polypeptidesor fragments thereof desirably greater than 10 amino acids in length(e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, or 440 amino acids or more), having a soluble endoglin biologicalactivity, and containing a sequence that has at least 70% sequenceidentity (e.g., at least 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to RVRWTVTC(SEQ ID NO: 1) or VRWTVTC (SEQ ID NO: 2) (see, FIGS. 1, 2A, and 3A-B). Asoluble endoglin polypeptide may contain at least 444 amino acids andhave a soluble endoglin biological activity. Desirably, the solubleendoglin polypeptide has a soluble endoglin biological activity andincludes a sequence that is at least 85% identical (e.g., at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) toSEQ ID NO: 3 (FIG. 1) (when an equal number of amino acids in thesoluble endoglin polypeptide are compared to the same number of aminoacids in SEQ ID NO: 3) or a sequence that is at least 70% identical(e.g., at least 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 2. In onenon-limiting example, the isolated soluble endoglin polypeptide has asoluble endoglin biological activity and includes or consists of theamino acid sequence of SEQ ID NO: 3. In another example, a solubleendoglin polypeptide has a soluble endoglin biological activity andcontains a sequence that is at least 70%, desirably at least 95%, 96%,97%, 98%, 99%, or 100% identical, to SEQ ID NO: 1 or SEQ ID NO: 2 at theC-terminus.

The invention also provides fragments of the above described solubleendoglin polypeptides that have a soluble endoglin biological activity.These fragments may exclude the signal sequence encoded by amino acids1-26 of SEQ ID NO: 3 (see FIG. 1). Fragments of the soluble endoglinpolypeptide have a soluble endoglin biological activity, contain atleast 10 contiguous amino acids (e.g., at least 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, or 440 amino acids or more), andcontain a sequence that has at least 70% sequence identity (e.g., atleast 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 2.

The provided soluble endoglin polypeptides and fragments may alsocontain additional N- and/or C-terminal sequences. For example,additional N- and/or C-terminal sequences may be added to a solubleendoglin polypeptide, having a soluble endoglin biological activity andcontaining a sequence is at least 85% identical (e.g., at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity) toSEQ ID NO: 3, or a fragment thereof, that has a soluble endoglinbiological activity. The additional N- and/or C-terminal sequences maybe at least 5 contiguous amino acids (e.g., at least 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 250, 300, 350, 400, 450, 500,550, or 600 amino acids). Examples of additional N— and/or C terminalsequences that may be covalently attached to a soluble endoglinpolypeptide or fragment are described below.

The soluble endoglin polypeptides or fragments provided have a solubleendoglin biological activity, e.g., one or more biological activityselected from, but not limited to: the ability to bind to TGF-β1, theability to bind to TGF-β2, the ability to bind to activin-A, the abilityto bind to BMP-2, the ability to bind to BMP-7 or BMP-9, the ability tobind TGF-β receptor I, the ability to bind to TGF-β receptor II, theability to bind BMP receptor II, the ability to reverse or inhibitangiogenesis, the ability to reduce or inhibit Smad 2/3-dependenttranscriptional activation, and the ability to inhibit eNOS activation.The soluble endoglin polypeptides or fragments desirably act asantagonists of endoglin or endoglin signaling pathways, to create adeficiency of TGF-β and other known ligands by acting as a physiologicalsink to bind ligands, and decrease TGF-β signaling in cells (e.g., anendothelial cell).

Soluble endoglin polypeptides useful in the methods of the inventioninclude any soluble endoglin polypeptide having a soluble endoglinbiological activity, containing a sequence that is at least 70%identical (e.g., at least 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to thesequence of SEQ ID NO: 2, or any homologs, fragments, derivatives, oranalogs thereof that have a soluble endoglin biological activity.

Soluble endoglin polypeptides, fragments, derivatives, and analogs ofthe invention can be produced by any of a variety of methods for proteinproduction known in the art, such as purification of naturally occurringsoluble endoglin polypeptides (e.g., from the placenta), products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, fungus, higher plant, insect, and mammalian cells.In one example, a soluble endoglin polypeptide of the invention isproduced by recombinant DNA methods by inserting a DNA sequence encodinga soluble endoglin polypeptide, or a fragment, derivative, or analogthereof, into a recombinant expression vector and expressing the DNAsequence under conditions promoting expression. General techniques fornucleic acid manipulation are described, for example, by Sambrook etal., in “Molecular Cloning: A Laboratory Manual,” 2nd Edition, ColdSpring Harbor Laboratory press, 1989; Goeddel et al., in “GeneExpression Technology Methods in Enzymology,” Academic Press, San Diego,Calif., 1990; Ausubel et al., in “Current Protocols in MolecularBiology,” John Wiley & Sons, New York, N.Y., 1998; Watson et al.,“Recombinant DNA,” Chapter 12, 2nd edition, Scientific American Books,1992; and other laboratory textbooks. The DNA encoding soluble endoglinpolypeptide or a fragment, derivative, or analog of the invention can beoperably linked to suitable transcriptional or translational regulatoryelements derived from mammalian, viral, or insect genes. Such regulatoryelements include a transcriptional promoter, an optional operatorsequence to control transcription, a sequence encoding suitable mRNAribosomal binding sites and sequences, which control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants may additionally beincorporated.

Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts can be found, for example,in “Cloning Vectors: A Laboratory Manual,” Elsevier, N.Y., 1985.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. The expression construct can be introduced for transient expressionof the protein or stable expression by selecting cells using aselectable marker in order to generate a stable cell line that expressesthe protein continuously. A variety of methods for introducing nucleicacids into host cells are known in the art, including, but not limitedto, electroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent).

Suitable host cells for expression of soluble endoglin polypeptide ofthe invention from recombinant vectors include prokaryotes, and fungal,mammalian, or insect cells.

In one embodiment, for recombinant expression of the full-length solubleendoglin, a nucleic acid encoding amino acids 1-444 (as shown in FIG. 1)are included in the construct used for expression and purification ofthe protein. In another embodiment, the soluble endoglin undergoespost-translation modification, including but not limited toglycosylation and s-nitrosylation and oxidation of a cysteine residueduring expression and purification. S-nitroslyation of soluble endoglinprevents its dimerization and may modulate its biological activity.

In addition, soluble endoglin polypeptides, fragments, derivatives, oranalogs of the invention may be expressed in transgenic mammals (e.g.,mice and bovines) such that the soluble endoglin is released into themilk of the transgenic animals. For example, the pBC1 Milk ExpressionVector Kit (Genzyme Transgenics Corporation and Invitrogen Corporation)provides a milk expression vector which features a casein expressionpromoter upstream of restriction site sequences (for insertion of theexpressed transgene). Methods of making transgenic animals and thepurification of transgenically-expressed soluble proteins from the milkof such animals are known in the art.

Purified soluble endoglin polypeptide, or biologically active fragments,derivatives, or analogs of the invention, are prepared by culturingsuitable host/vector systems to express the recombinant proteins. As asecreted protein, soluble endoglin polypeptide is likely to be releasedand can then be purified from culture media or cell extracts.

In one example, supernatants from systems which secrete recombinantprotein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit, and then purified.

In addition to the methods employing recombinant DNA, soluble endoglinpolypeptides or fragments of the invention can be purified from sourcesthat naturally produce this form of the protein. Examples of thesesources include any mammalian tissue or cells, such as placentaltissues. The soluble endoglin polypeptide from these sources can bepurified and concentrated using any of the methods known in the art ordescribed herein.

After purification, soluble endoglin polypeptide or fragment of theinvention may be exchanged into different buffers and/or concentrated byany of a variety of methods known in the art, including, but not limitedto, filtration and dialysis. The purified soluble endoglin polypeptideor fragment is preferably at least 80% or 85% pure, more preferably atleast 90% or 95% pure, and most preferably at least 98% pure. Regardlessof the exact numerical value of the purity, the soluble endoglinpolypeptide is preferably sufficiently pure for use as a pharmaceuticalproduct.

Soluble endoglin polypeptides, or fragments, derivatives, or analogs ofthe invention, can also be produced by chemical synthesis (e.g., by themethods described in “Solid Phase Peptide Synthesis,” 2nd ed., ThePierce Chemical Co., Rockford, Ill., 1984). Modifications to theprotein, such as those described below, can also be produced by chemicalsynthesis.

Modifications to Soluble Endoglin Polypeptides

The invention encompasses soluble endoglin polypeptides, or fragments,derivatives, or analogs having a soluble endoglin biological activity,which are modified during or after synthesis or translation.

Modifications to the sequence of a soluble endoglin polypeptide of theinvention (e.g., polypeptides containing the sequence of SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3) may be made by deletion, addition, oralteration of the amino acids incorporated into the soluble endoglinpolypeptide during translation without destroying the activity of theprotein. Such sequence modifications can be made to improve expression,stability, solubility, cellular uptake, or biological activity of theprotein in the various expression systems. In one example, thepost-translational modification is the S-nitrosylation of a cysteine inthe soluble endoglin protein. For example, a mutation can increase ordecrease the binding of soluble endoglin polypeptide to TGF-β (onesoluble endoglin biological activity). Generally, substitutions are madeconservatively and take into consideration the effect on one or moresoluble endoglin biological activity. Mutations, deletions, or additionsin nucleotide sequences constructed for expression of derivative oranalog proteins or fragments thereof must, of course, preserve thereading frame of the coding sequences and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructures such as loops or hairpins which would adversely affecttranslation of the mRNA.

Post-translational modifications may provide additional advantages suchas increased affinity; decreased off-rate; increased solubility,stability, and in vivo or in vitro circulating time of the polypeptide;or decreased immunogenicity; and include, for example, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, S-nitrosylation, disulfide bond formation,demethylation, formation of cysteine, formation of sulfonic, sulfenic,or sulfinic acid, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination (see, for example,Creighton, “Proteins: Structures and Molecular Properties,” 2nd Ed., W.H. Freeman and Co., N.Y., 1992; “Postranslational Covalent Modificationof Proteins,” Johnson, ed., Academic Press, New York, 1983; Seifter etal., Meth. Enzymol., 182:626-646, 1990; Rattan et al., Ann. N.Y. Acad.Sci., 663:48-62, 1992). Additionally, the soluble endoglin polypeptideof the invention may contain one or more non-classical amino acids.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4-diaminobutyric acid,α-amino-isobutyric acid, 4-aminobutyric acid, Abu, 2-amino-butyric acid,g-Abu, e-Ahx, 6-amino-hexanoic acid, Aib, 2-amino-isobutyric acid,3-amino-propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,α-alanine, fluoro-amino acids, designer amino acids such as α-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends, attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression.

In one preferred example, the post-translational modification isS-nitrosylation of a cysteine in the soluble endoglin protein.

As described above, the invention also includes chemically-modifiedderivatives of the presently described soluble endoglin polypeptides,which may provide additional advantages such as increased solubility,stability, and circulating time of the polypeptide, or decreasedimmunogenicity (see, e.g., U.S. Pat. No. 4,179,337; incorporated byreference). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as, for example, polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol, and the like. The soluble endoglinpolypeptide or fragment of the invention may be modified at randompositions within the molecule, or at predetermined positions within themolecule and may include one, two, or three or more attached chemicalmoieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). As noted above,the polyethylene glycol may have a branched structure. Branchedpolyethylene glycols are described, for example, in U.S. Pat. No.5,643,575 (incorporated by reference); Morpurgo et al., Appl. Biochem.Biotechnol. 56:59-72, 1996; Vorobjev et al., Nucleosides Nucleotides18:2745-2750, 1999; and Caliceti et al., Bioconjug. Chem. 10:638-646,1999, the disclosures of each of which are incorporated by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the soluble endoglin polypeptide or fragment of theinvention with consideration of effects on functional or antigenicdomains of the protein. There are a number of attachment methodsavailable to those skilled in the art, e.g., EP 0401384 (coupling PEG toG-CSF; herein incorporated by reference), see also Malik et al., Exp.Hematol. 20:1028-1035, 1992 (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues, glutamic acid residues, and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or a lysine residue. The number of polyethylene glycolmoieties attached to each polypeptide or fragment of the invention(i.e., the degree of substitution) may also vary. For example, thepegylated soluble endoglin polypeptide or fragment may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution may vary within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7,6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18,17-19, or 18-20 polyethylene glycol moieties per polypeptide or fragmentmolecule. Methods for determining the degree of substitution arediscussed, for example, in Delgado et al., Crit. Rev. Ther. Drug CarrierSys., 9:249-304, 1992.

The soluble endoglin polypeptides of fragments of the invention may alsobe modified with a detectable label, including, but not limited to, anenzyme, prosthetic group, fluorescent material, luminescent material,bioluminescent material, radioactive material, positron emitting metal,nonradioactive paramagnetic metal ion, and affinity label for detectionand isolation of a soluble endoglin target. The detectable substance maybe coupled or conjugated either directly to the polypeptides of theinvention or indirectly, through an intermediate (such as, for example,a linker known in the art) using techniques known in the art.Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, oracetylcholinesterase; non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin; non-limitingexamples of suitable fluorescent materials include biotin,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin;an example of a luminescent material includes luminol; non-limitingexamples of bioluminescent materials include luciferase, luciferin, andaequorin; and examples of suitable radioactive material include aradioactive metal ion, e.g., alpha-emitters or other radioisotopes suchas, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹¹⁵mIn, ¹¹³mIn, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ⁸⁶R, ¹⁸⁸Re,¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ₅₇Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr,⁵⁴Mn, ⁷⁵Se, and tin (¹¹³Sn, ¹¹⁷Sn). The detectable substance may becoupled or conjugated either directly to the soluble endoglinpolypeptide of fragment of the invention or indirectly, through anintermediate (such as, for example, a linker known in the art) usingtechniques known in the art. See, for example, U.S. Pat. No. 4,741,900(incorporated by reference) for metal ions, which can be conjugated toone of the provided soluble endoglin polypeptides or fragments for usein diagnostics according to the present invention.

The soluble endoglin polypeptide or fragment of the invention can alsobe modified by conjugation to another protein or therapeutic compound.Such conjugation can be used, for example, to enhance the stability orsolubility of the protein, to reduce the antigenicity, or to enhance thetherapeutic effects of the protein. A preferred fusion protein comprisesa heterologous region from immunoglobulin (e.g., all or part of the Fcregion) that is useful to solubilize proteins (see, EP-A 0232 262).

A soluble endoglin polypeptide or fragment of the invention may beconjugated to a therapeutic moiety such as a cytotoxin, e.g., acytostatic or cytocidal agent, a chemotherapeutic agent, aradiotherapeutic agent, or a radioactive metal ion, e.g., alpha-emitterssuch as, for example, ²¹³Bi, or other radioisotopes such as, forexample, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y,¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁵¹Cr, ₅₄Mn, ⁷⁵Se, ¹¹³Sn,⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include, but are not limited to, paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease,RNAse, and puromycin and fragments, variants or homologs thereof.

Additional therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, and 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thiotepa, chlorambuil, melphalan, carmustine (BSNU),lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Techniques known in the art may be applied to label soluble endoglinpolypeptides or fragments of the invention. Such techniques include, butare not limited to, the use of bifunctional conjugating agents (see,e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;4,994,560; and 5,808,003; the relevant disclosures of each of which arehereby incorporated by reference in its entirety) and direct couplingreactions (e.g., Bolton-Hunter and Chloramine-T reaction).

Expression and Activity Assays for Soluble Endoglin Polypeptides

The expression levels of soluble endoglin (e.g., protein or mRNA) andthe activity of the soluble endoglin polypeptides, fragments,derivatives, or analogs described herein may be measured using knownmethods (see, e.g., WO 07/143,023 and WO 08/030,283, incorporated byreference).

The following methods may be used to evaluate the biological activity ofa soluble endoglin polypeptide, fragment, derivative, or analog of theinvention and may also be used to determine ability of the compounds ofthe invention to decrease or increase soluble endoglin expression (e.g.,protein or mRNA) or a soluble endoglin polypeptide biological activityin a subject.

To measure the ability of a compound to alter soluble endoglinpolypeptide expression in a subject, blood serum from a subject may bemeasured for levels of soluble endoglin polypeptide of the invention,using methods such as ELISA, western blotting, or immunoassays usingspecific antibodies. Blood serum from the subject can also be measuredfor levels of TGF-β1, TGF-β3, activin-A, BMP2, BMP7, or any proteinligand known to bind to soluble endoglin polypeptide. Methods used tomeasure serum levels of these additional proteins include ELISA, westernblotting, or immunoassays using specific antibodies. In addition, invitro angiogenesis assays can be used to determine if the subject'sblood has converted from an anti-angiogenic state to a pro-angiogenicstate. Blood, serum, or urine samples from the subject can also bemeasured for levels of nucleic acids or polypeptides encoding eNOS,TFG-β1, TGF-β3, activin-A, BMP2, BMP7, or a soluble endoglin polypeptideof the invention. There are several art-known methods to assay for geneexpression. Some examples include the preparation of RNA from the bloodsamples of the subject and the use of the RNA for northern blotting,PCR-based amplification, or RNAse protection assays. A decrease in thelevel of one of more of TGF-β1, TGF-β3, activin-A, BMP2, and BMP7indicates that an administered soluble endoglin polypeptide, fragment,derivative, or analog of the invention has a soluble endoglinpolypeptide biological activity or an administered compound has theeffect of increasing soluble endoglin polypeptide biological activity orexpression levels. Conversely, an increase in the level of one of moreof TGF-β1, TGF-β3, activin-A, BMP2, and BMP7 indicates a decrease insoluble endoglin polypeptide biological activity or expression levels.In addition, the above methods for the measurement of the expression ofa soluble endoglin (e.g., polypeptide or nucleic acid) can also beperformed to measure the expression of a soluble endoglin of theinvention in a cell (e.g., a cell cultured in vitro).

The ability of a soluble endoglin polypeptide, fragment, derivative, oranalog of the invention to inhibit angiogenesis may be determined usingthe assays described in U.S. Patent Application Publication No.2006/0067937 and PCT Publication No. WO 06/034507 (each incorporated byreference). In these experiments, Evans blue avidly binds to albumin andis used to quantify in vivo permeability in animals and humans (Green etal., J. Lab. Clin. Med. 111:173-183, 1988). Briefly, Balb-C mice areinjected through the retro-orbital venous plexus with 1×10⁸ pfu ofadenovirus expressing GFP or soluble endoglin polypeptide and amicrovascular permeability assay is performed 48 hours later. Mice areanesthetized by IP injection of 0.5 mL Avertin. 100 mL of 1% Evans bluedye (in PBS) is injected into the tail vein. 40 minutes later, mice areperfused via heart puncture with PBS containing 2 mM EDTA for 20minutes. Organs (brain, lung, liver, and kidney) are harvested andincubated in formamide for 3 days to elute Evans blue dye. The opticaldensity of the formamide solution is measured using 620 nm wavelength.

The ability of a soluble endoglin polypeptide, fragment, derivative, oranalog of the invention to decrease microvascular reactivity may bedetermined using miscrovascular reactivity experiments as described inMaynard et al, J. Clin. Invest. 111:649-658, 2003 using rat renalmicrovessels (70-170 μm internal diameter). In these experiments, therelaxation properties of kidney microvessels are examined afterpre-contraction of the microvessels with U46619 (thromboxane agonist) to40-60% of their baseline diameter at a distending pressure of 40 mmHgOnce the steady-state tone was reached, the responses to variousreagents such as TGF-β1 or TGF-β3 or VEGF may be examined in astandardized order. In these assays, all drugs are appliedextraluminally.

The ability of a soluble endoglin polypeptide, fragment, derivative, oranalog of the invention to inhibit eNOS activation are described in WO08/030,283 (incorporated by reference). The ability of a solubleendoglin polypeptide, fragment, derivative, or analog to bind a TGF-βfamily member (e.g., TGF-β1, TGF-β3, activin-A, and BMP), to bind aTGF-β receptor (e.g., TβRI and TβRII), to block binding of TGF-β1 toTβRII, and to inhibit TGF-β signaling pathways are known in the art andare described in WO 08/030,283 (incorporated by reference). Additionalassays for soluble endoglin biological activity include reporter geneassays for downstream signaling proteins, such as Smad2/3-dependenttranscription. Binding assays are also well known in the art. Forexample, a BIAcore instrument can be used to determine the bindingconstant of a complex between two proteins. The dissociation constantfor the complex can be determined by monitoring changes in therefractive index with respect to time as buffer is passed over the chip(O'Shannessy et al., Anal. Biochem. 212:457-468, 1993; Schuster et al.,Nature 365:343-347, 1993). Other suitable assays for measuring thebinding of one protein to another include, for example, immunoassayssuch as enzyme-linked immunoabsorbent assays (ELISA) andradioimmunoassays (RIA), or determination of binding by monitoring thechange in the spectroscopic or optical properties of the proteinsthrough fluorescence, UV absorption, circular dichroism, or nuclearmagnetic resonance (NMR). Additional examples of such assays are knownin the art.

Therapeutic Applications

The present invention features methods and compositions for treating ordecreasing the likelihood of developing a soluble endoglin-mediateddisorder or a soluble endoglin-preventive disorder (as describedherein).

Soluble Endoglin-Mediated Disorders

The invention provides compositions and methods for treating or reducingthe likelihood of developing a soluble endoglin-mediated disorder in asubject (e.g., a human). A soluble endoglin-mediated disorder is anydisorder where increased soluble endoglin polypeptide or an increase ina soluble endoglin polypeptide biological activity is implicated in thepathogenesis or development of the disorder. Non-limiting examples ofsoluble endoglin-mediated disorders include pregnancy-relatedhypertensive disorders (e.g., pre-eclampsia, eclampsia, gestationalhypertension, abruption placenta, pregnancy with intrauterine growthrestriction, chronic hypertension, HELLP syndrome, pregnancy with asmaller-for-gestational age (SGA) infant), pulmonary hypertension,cancer (where soluble endoglin levels are increased) and malaria.Additional examples include Alzheimer's disease, cerebral arteriovenousmalformations, and left ventricular dysfunction. In addition, patientswith diabetes having retinopathy and/or a high probability of 10-yearcardiovascular risk and patients with diabetes and hypertension who havethree or more damaged target organs (e.g., heart, vessels, and kidney)are also included in some embodiments

Compositions for treating, ameliorating, preventing, or reducing thelikelihood of developing a soluble endoglin-mediated disorder preferablycontain a compound that can decrease the expression levels (polypeptide,small molecule, or mRNA) or a biological activity of a soluble endoglinpolypeptide (as described herein). Preferably, the compound is apurified antibody or antibody-binding fragment that specifically bindsto the soluble endoglin polypeptide (e.g., specifically binds to anepitope containing a sequence that is at least 70%, 71%, 72%, 73%, 74%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2, or a fragment thereof). The compound may alsobe an antisense nucleobase oligomer, an inhibitory nucleic acid (e.g., adsRNA used to mediate RNA interference or a microRNA), or a smallmolecule (e.g., a compound capable of decreasing the expression level(polypeptide or mRNA) or a biological activity of a soluble endoglinpolypeptide of the invention). Exemplary antisense nucleobase oligomers,antibodies, and compounds useful for the treatment of a solubleendoglin-mediated disorder are described below.

Antibodies, or antibody-binding fragments, thereof that specificallybind to soluble endoglin polypeptides or fragments described herein maybe used to treat or prevent the development of a solubleendoglin-mediated disorder in a subject. Desirably, antibodies, orantibody-binding fragments thereof, specifically bind to an epitope thatcontains a sequence at least 70% identical (e.g., at least 71%, 72%,73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical) to SEQ ID NO: 2, or a fragment thereof (e.g., 3amino acids, 4 amino acids, 5 amino acids, or 6 amino acids). In oneexample, the antibody competes with C-5144 antibody for binding tosoluble endoglin. The antibodies or antibody-binding fragments describedherein may act as an antagonist or an agonist of a soluble endoglinpolypeptide or fragment of the invention or may act as an antagonist oran agonist of a receptor that binds a soluble endoglin polypeptide orfragment of the invention. These antibodies and antibody fragments canbe used to neutralize the activity of the soluble endoglin polypeptideor fragment blocking the binding of TGF-β1, TGF-β1, activin-A, BMP2, orBMP7. Soluble endoglin can also associate with its cell surfacecounterpart and can interact with the same receptors that the cellsurface form of Eng is able to bind to and/or regulate including but notlimited to receptors such as Alk-1, Alk-5, BMPR2. It is also possiblethat soluble endoglin may cause autonomous inhibition or activation ofthese receptors, independently of any ligand. Antibodies directed to thesoluble endoglin of the invention can be used to neutralize any of theabove activities of soluble endoglin.

Methods for the preparation and use of antibodies or antibody-bindingfragments thereof for therapeutic purposes are described in severalpatents including U.S. Pat. Nos. 6,054,297; 5,821,337; 6,365,157; and6,165,464; U.S. Patent Application Publication No. 2006/0067937; and PCTPublication No. WO 06/034507 (each incorporated by reference).Antibodies can be polyclonal, monoclonal, chimeric, bispecific, or a Fabfragment; monoclonal humanized antibodies are preferred.

The antibodies may be administered to the subject in a therapeuticallyeffective amount. Preferably, the antibodies are administeredparenterally or intravenously by continuous infusion. The dose anddosage regimen depends upon the severity of the solubleendoglin-mediated disorder, and the overall health of the subject. Theamount of the antibody administered is typically in the range of about0.001 to about 10 mg/kg of subject weight, preferably 0.01 to about 5mg/kg of subject weight.

For parenteral administration, the antibodies are formulated in a unitdosage injectable form (solution, suspension, emulsion) in associationwith a pharmaceutically acceptable parenteral vehicle. Such vehicles areinherently nontoxic, and non-therapeutic. Examples of such vehicles arewater, saline, Ringer's solution, dextrose solution, and 5% human serumalbumin. Nonaqueous vehicles such as fixed oils and ethyl oleate mayalso be used. Liposomes may also be used as carriers. The vehicle maycontain minor amounts of additives such as substances that enhance theisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies are typically formulated in such vehicles at concentrationsof about 1 mg/mL to 10 mg/mL.

Antisense nucleobase oligomers that reduce or inhibit the expression ofa nucleic acid encoding a soluble endoglin polypeptide or fragment asdescribed herein may be used to treat or prevent the development of asoluble endoglin-mediated disorder in a subject. By binding to thecomplementary nucleic acid sequence (e.g., a sequence that is at least70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence encodingSEQ ID NO: 2) (the sense or coding strand), antisense nucleobaseoligomers are able to inhibit protein expression presumably through theenzymatic cleavage of the RNA strand by RNAse H. Preferably theantisense nucleobase oligomer is capable of reducing soluble endoglinpolypeptide expression in a cell that expresses increased levels ofsoluble endoglin polypeptide as described herein. Preferably thedecrease in the soluble endoglin polypeptide expression is at least 10%relative to cells treated with a control oligonucleotide, preferably 20%or greater, more preferably 40%, 50%, 60%, 70%, 80%, 90%, or greater.Methods for selecting and preparing antisense nucleobase oligomers arewell known in the art. Methods for assaying levels of protein expressionare also well known in the art and include western blotting,immunoprecipitation, and ELISA.

The present invention also features the use of RNA interference (RNAi)to inhibit expression of a soluble endoglin polypeptide or a fragmentthereof. RNA interference (RNAi) is a recently discovered mechanism ofpost-transcriptional gene silencing (PTGS) in which double-stranded RNA(dsRNA) corresponding to a gene or mRNA of interest is introduced intoan organism resulting in the degradation of the corresponding mRNA. Inthe RNAi reaction, both the sense and anti-sense strands of a dsRNAmolecule are processed into small RNA fragments or segments ranging inlength from 16 to 23 nucleotides (nt) and have 2-nucleotide 3′ tails.Alternatively, synthetic dsRNAs, which are 21 to 23 nt in length andhave 2-nucleotide 3′ tails can be synthesized, purified, and used in thereaction. These 21 to 23 nt dsRNAs are known as “guide RNAs” or “shortinterfering RNAs” or “siRNAs.”

The siRNA duplexes then bind to a nuclease complex composed of proteinsthat target and destroy endogenous mRNAs having homology to the siRNAwithin the complex. Although the identity of the proteins within thecomplex remains unclear, the function of the complex is to target thehomologous mRNA molecule through base pairing interactions between oneof the siRNA strands and the endogenous mRNA. The mRNA is then cleavedapproximately 12 nt from the 3′ terminus of the siRNA and degraded. Inthis manner, specific genes can be targeted and degraded, therebyresulting in a loss of protein expression from the targeted gene. siRNAscan also be chemically synthesized or obtained from a company thatchemically synthesizes siRNAs (e.g., Dharmacon Resesarch Inc.,Pharmacia, or ABI).

The specific requirements and modifications of dsRNA are known in theart and are described, for example, in PCT Publication No. WO 01/75164and in U.S. Patent Application Publication No. 20060067937 and PCTPublication No. WO 06/034507 (each incorporated herein by reference).

The administration of the above antibodies, inhibitory nucleic acids,and small compounds may be combined with methods to decrease sFlt-1levels or to increase VEGF or P1GF levels, or decrease s-Flt-1 levels asdescribed in PCT Publication Nos. WO 04/008947, WO 07/143,023, and WO08/030,283, and U.S. Patent Publication Nos. 20040126828 and 20050170444(each incorporated by reference). In addition, any compound thatincreases the level or biological activity of TGF-13, eNOS, and/or PGI2are useful in the methods of the invention. Purified TGF-β familyproteins include any protein with an amino acid sequence that ishomologous, more desirably, substantially identical to the amino acidsequence of TGF-β1 (Cat #240-B-002) and human TGF-β3 (Cat #243-B3-002)from R & D Systems, M N. Preferred TGF-β family proteins useful in themethods of the invention will have the ability to bind soluble endoglinpolypeptide. Cyclosporin may also be used at a dosage of 100-200 mgtwice a day to stimulate TGF-β1 production. NOS activity may beincreased using purified NOS (e.g., eNOS), nucleic acids encoding eNOS,statins, vanadate, hepatocyte growth factor, phosphinositide 3-kinase(PI3K), Akt, VEGF, TGF-β1, or any other compound that increases eNOS Ser1177 phosphorylation or Thr 495 dephosphorylation or both. Compounds theincrease the biological activity of NOS can be administered incombination with L-arginine or a nitric oxide donor (e.g., sodiumnitroprusside, nitroglycerin, isosorbidmononitrate, and isosorbodinitrate). Compounds that increase the level or activity of PGI2 mayalso be administered (e.g., PGI2 mimetics, iloprast, cicaprost, andaspirin).

Any of the above compounds that can be used to treat a solubleendoglin-mediated disorder may be used alone or in combination with oneor more additional agents. For example, one of more of the abovedescribed compounds may be administered in combination with any otherstandard pre-eclampsia or eclampsia therapy, e.g., those therapiesdescribed in U.S. Patent Application Publication Nos. 2004/0126828,2005/0025762, 2005/0170444, 2006/0067937, and 2007/0104707, and PCTPublication Nos. WO 04/008946, WO 05/077007, WO 06/034507, WO07/143,023, and WO 08/030,283 (each incorporated by reference). It willbe understood by the skilled artisan that any combination of any ofthese agents can be used for this purpose. For example, an antibody thatspecifically binds to the soluble endoglin polypeptide of the inventionor an antibody-binding fragment thereof can be administered incombination with VEGF. In another example, a compound that increasesTGF-β1 levels or activity can be administered in combination with acompound that increases VEGF or P1GF in order to target both theendoglin and the VEGF pathway. Alternatively, a combination ofantibodies or antibody-binding fragments thereof against both solubleendoglin polypeptide of the invention and sFlt-1 may be used eitherdirectly or in an ex vivo approach (e.g., using a column that is linedwith anti-soluble endoglin polypeptide and anti-s-Flt-1 antibodies andcirculating the subject's blood through the column). Any of thesecombinations can further include the administration of a compound thatincreases NOS levels or activity, preferably eNOS, in order to regulatethe pathway downstream of the respective receptors.

In addition, the invention provides for the use of any chronichypertension medications used in combination with any of the therapeuticmethods described above. For example, medications used for the treatmentof hypertension during pregnancy include methyldopa, hydralazinehydrochloride, or labetalol may be used in combination with an antibodythat specifically binds soluble endoglin polypeptides of the invention.For each of these medications, modes of administration and dosages aredetermined by the physician and by the manufacturer's instructions.

Soluble Endoglin-Preventive Disorders

The invention further provides compositions and methods for treating orpreventing the likelihood of developing a soluble endoglin-preventivedisorder in a subject including but not limited to a disorder where adecrease in the soluble endoglin levels (protein or mRNA) or the solubleendoglin biological activity has been detected or implicated in thepathogenesis of the disorder or where specific factors, such as TGF β orevents (e.g. fibrosis, angiogenesis, immune activation) that can beinhibited or modulated by soluble endoglin, which contribute to thedisorder. Non-limiting examples include fibrotic disorders of internalorgans and the scarring of skin where TGF-β is a significantcontributor, disorders characterized by excessive angiogenesis (e.g.hemangiomas, pulmonary capillary hemagiomatosis) or abnormal growth ofblood vessel such as cancer (e.g., cancer of the breast, prostate,colon, lung, head and neck, liver, kidney, renal system, andendometrium) and inflammatory and immune disorders (e.g. excessive TGF-βmay be related to autoimmune diseases).

The term cancer embraces a collection of malignancies with each cancerof each organ consisting of numerous subsets. Typically, at the time ofcancer diagnosis, “the cancer” consists in fact of multiplesubpopulations of cells with diverse genetic, biochemical, immunologic,and biological characteristics. Benign or malignant growths of cancerare referred to as tumors. The tumor can be a solid tumor or a non-solidor soft tissue tumor. Examples of soft tissue tumors include leukemia(e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adultacute lymphoblastic leukemia, mature B-cell acute lymphoblasticleukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, orhairy cell leukemia), or lymphoma (e.g., non-Hodgkin's lymphoma,cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors can befurther separated into those of epithelial origin and those ofnon-epithelial cell origin. Examples of epithelial cell solid tumorsinclude tumors of the gastrointestinal tract, colon, breast, prostate,lung, kidney, liver, pancreas, ovary, head and neck, oral cavity,stomach, duodenum, small intestine, large intestine, anus, gall bladder,labium, nasopharynx, skin, uterus, endometrium, male genital organ,urinary organs, bladder, and skin. Solid tumors of non-epithelial origininclude sarcomas, brain tumors, and bone tumors. While the methods ofthe invention can be used to treat any tumor or tumor metastasis wheresoluble endoglin can be used to modulate or inhibit the occurrence,growth, or metastasis of the tumor, a soluble endoglin polypeptide,fragment, derivative, or analog of the invention, a nucleic acidencoding a soluble endoglin polypeptide of the invention, or a smallmolecule that increases the expression level (polypeptide or mRNA) or abiological activity of a soluble endoglin polypeptide of the inventionis preferably used for the treatment or prevention of cancers that haveincreased TGF-β levels or biological activity, particularly TGF-β1 orTGF-β3, or tumors that have angiogenic activity.

Of particular importance to the present invention are subjects diagnosedwith and/or treated for a primary tumor, including prophylactictreatment of at-risk subjects, not yet diagnosed with metastatic diseaseor determined to lack metastatic disease, and those subjects otherwisepredisposed to developing metastatic disease. The methods of theinvention can be used to prevent the occurrence or re-occurrence ofmetastatic disease. Also included are subjects who have undergonetreatment of metastasis or a possible metastasis in order to prevent orreduce metastatic disease. The methods of the invention can be usedbefore, during, or after additional therapies to treat the primarytumor, the metastases, or the risk of either.

Compositions for treating or preventing a soluble endoglin-preventivedisorder preferably contain any of the purified soluble endoglinpolypeptides, fragments, derivatives, or analogs described above or anucleic acid encoding such a soluble endoglin polypeptide (e.g., SEQ IDNO: 4). Compositions for treating or reducing the likelihood ofdeveloping a soluble endoglin-preventive disorder may also contain anagent that can increase the expression levels (polypeptide or mRNA) or abiological activity of a soluble endoglin polypeptide as describedabove. Non-limiting examples of soluble endoglin polypeptides andnucleic acids encoding these soluble endoglin polypeptides are describedabove.

Nucleic acids encoding soluble endoglin polypeptides, or fragments,derivatives, or analogs thereof (as described above) can also be used inmethods for treating or reducing the likelihood of developing a solubleendoglin-preventive disease. The nucleic acids encoding a solubleendoglin polypeptide, fragment, derivative, or analog of the inventionmay be obtained using routine procedures in the art, e.g., recombinantDNA and PCR amplification For any of the nucleic acid applicationsdescribed herein, standard methods for administering nucleic acids canbe used. Examples are described in U.S. Patent Application PublicationNo. 2006/0067937 and PCT Publication No. WO 06/034507 (each incorporatedby reference).

Delivery of nucleic acids to endothelial cells can be used in thepresent invention for the delivery of nucleic acids encoding solubleendoglin polypeptides, fragments, derivatives, or analogs of theinvention. These general techniques are described in U.S. Pat. Nos.5,830,879 and 6,258,727 (each incorporated by reference).

Compounds useful in the methods of the invention will increase thepresently described soluble endoglin polypeptide or nucleic acid (e.g.,mRNA) expression levels, or increase at least one biological activity ofa soluble endoglin polypeptide by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or 95% or more. Solubleendoglin polypeptide expression level and biological activity can bedetermined using the assays described above.

The invention also includes mimetics, based on modeling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size, and charge characteristics. Followingidentification of a therapeutic compound, suitable modeling techniquesknown in the art can be used to study the functional interactions anddesign mimetic compounds which contain functional groups arranged insuch a manner that they could reproduce those interactions. Thedesigning of mimetics to a known pharmaceutically active compound is aknown approach to the development of pharmaceuticals based on a leadcompound. This might be desirable where the active compound is difficultor expensive to synthesize or where it is unsuitable for a particularmethod of administration, e.g., peptides are not well suited as activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis, andtesting may be used to avoid randomly screening a large number ofmolecules for a target property. The mimetic or mimetics can then bescreened to see whether they increase the presently described solubleendoglin polypeptide or nucleic acid levels or soluble endoglinpolypeptide biological activity, and further optimization ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

The soluble endoglin can also be delivered to the subjects usingautologous or non-autologous stem cells (e.g., mesenchymal stem cells)to express the soluble endoglin protein. The transgenic stem cells canbe prepared and delivered to the subject using techniques known in theart.

For the treatment of cancer, the above described compositions may beadministered in conjunction (e.g., before, after, during) withadditional cancer therapies to prevent or reduce tumor growth ormetastasis. Treatment therapies include but are not limited to surgery,radiation therapy, chemotherapy, immune therapy (e.g., cytokines,cancer-specific antibodies, interferons, and biologics), differentiatingtherapy, anti-angiogenic therapy, hormone therapy, or hyperthermia.

It should be noted that while soluble endoglin may inhibit TGF-βsignaling or activity it may have the opposite effect in other celltypes or with other members of TGF-β superfamily (e.g., BMPs orActivins) and in those cases, inhibitors of soluble endoglin, such asthose described herein are useful.

In addition to the above compositions, one or more additional cancertherapeutic (e.g., one or more of an angiogenesis inhibitor, ananti-proliferative compound, or a chemotherapeutic compound) may beco-administered to a subject for treating or preventing the developmentor a soluble endoglin-preventive disorder. For example, the solubleendoglin polypeptides, fragments, derivatives, or analogs describedherein may be formulated alone or in combination with any additionalcancer therapies in a variety of ways that are known in the art. Suchadditional cancer therapies can be administered before, during, or afterthe administration of the soluble endoglin polypeptides, fragments,derivatives, or analogs of the invention. Non-limiting examples ofangiogenesis inhibitors, anti-proliferative compounds, andchemotherapeutic compounds are described below.

Angiogenesis inhibitors, also known as anti-angiogenic agents, that maybe used in combination with any of the above soluble endoglinpolypeptides, fragments, derivatives, and analogs or soluble endoglinnucleic acids for treating a cancer include: an anti-angiogenic antibody(e.g., an antibody that binds VEGF-A or an antibody that binds a VEGFreceptor and blocks VEGF binding (e.g., anti-VEGF antibody and thosedescribed in U.S. Patent Publication Nos. 2003/0175271, 2005/0186208,2006/0030529, 2007/0025999, 2007/0036753, 2007/003654, 2007/0036755,2007/0036790, 2007/0071718, 2007/0071748, and 2007/0071749; eachincorporated by reference)), VEGF trap, soluble VEGF receptor (e.g.,sFlt1 and those described in U.S. Pat. Nos. 5,712,380; 5,861,484; and7,071,159; and U.S. Patent Publication Nos. 2003/0120038, 2005/0276808,and 2007/0037748; each incorporated by reference), endostatin,angiostatin, restin, tumstatin, TNP-470, 2-methoxyestradiol,thalidomide, antibodies that inhibit TGF-β biological activity, apeptide fragment of an anti-angiogenic protein, canstatin, arrestin, aVEGF kinase inhibitor (e.g., SU11248, PTK787, BAY 43-9006,1,5-diarylbenzimidazoles, and the inhibitors disclosed in U.S. Pat. Nos.6,448,277; 6,465,484; and 7,045,133; and U.S. Patent Publication Nos.2005/0085637, 2005/0234083, 2005/0288515, 2006/0135501, 2006/0160861,2006/0264425, and 2007/0015756; each incorporated by reference),CPTK787, SFH-1, an anti-angiogenic protein, thrombospondin-1, plateletfactor-4, interferon-α, an agent that blocks TIE-1 or TIE-2 signaling,an agent that blocks PIH12 signaling, an agent that blocks anextracellular vascular endothelial (VE) cadherin domain, an antibodythat binds to an extracellular VE-cadherin domain, tetracycline,penicillamine, vinblastine, cytoxan, edelfosine, tegafur or uracil,curcumin, green tea, genistein, resveratrol, N-acetyl cysteine,captopril, a COX-2 inhibitor, celecoxib, and rofecoxib. Preferredcombinations will include a soluble endoglin polypeptide, fragment,derivative, or analog, or a soluble endoglin nucleic acid in combinationwith a VEGF inhibitor or VEGF antagonist as described above (e.g.,bevacizumab, VEGF trap, sFlt1, or an antibody that specifically bindsVEGF).

The dosage of the angiogenesis inhibitor will depend on other clinicalfactors such as weight and condition of the human or animal and theroute of administration of the compound. For treating humans or animals,between approximately 0.5 mg/kg to 500 mg/kg body weight of theangiogenesis inhibitor can be administered. A more preferable range is 1mg/kg to 100 mg/kg body weight with the most preferable range being from2 mg/kg to 50 mg/kg body weight. Depending upon the half-life of theangiogenesis inhibitor in the particular animal or human, theangiogenesis inhibitor can be administered between several times per dayto once a week. The methods of the present invention provide for singleas well as multiple administrations, given either simultaneously or overan extended period of time.

In addition, the invention provides for the use of an anti-proliferativecompound used in combination with any of the soluble endoglinpolypeptides, fragments, derivatives, or analogs, or soluble endoglinnucleic acids of the invention for treating a cancer. Anti-proliferativecompounds that may be used include taxol, troglitazone, an antibody thatbinds basic fibroblast growth factor (bFGF), an antibody that bindsbFGF-saporin, a statin, an acetylcholinesterase (ACE) inhibitor,suramin, 17-beta-estradiol, atorvastatin, fluvastatin, lovastatin,pravastatin, simvastatin, cerivastatin, perindopril, quinapril,captopril, lisinopril, enalapril, fosinopril, cilazapril, ramipril, anda kinase inhibitor.

The dosage of the anti-proliferative compound depends on clinicalfactors such as weight and condition of the human or animal and theroute of delivery of the compound. In general, for treating humans oranimals, between approximately 0.1 mg/kg to 500 mg/kg body weight of theanti-proliferative compound can be administered. A more preferable rangeis 1 mg/kg to 50 mg/kg body weight with the most preferable range beingfrom 1 mg/kg to 25 mg/kg body weight. Depending upon the half-life ofthe anti-proliferative compound in the particular animal or human, thecompound can be administered between several times per day to once aweek. The methods of the present invention provide for single as well asmultiple administrations, given either simultaneously or over anextended period of time.

It should be noted that although each of the compounds is listed under aspecific category of compounds, these categories are not meant to belimiting in scope. Many of the compounds possess more than one activityand can therefore be included under more than one category.

For each of the compounds listed, all of the modes of administrationdescribed herein can be used. As some of the compounds described haveshown toxicity when administered orally or systemically, localadministration can also be used. In general, percent composition of thecompound will range from 0.05% to 50% weight for weight of compound tocoating material used.

Doses and Modes of Administration

Techniques and dosages for administration of the above describedcompositions useful for the treatment or amelioration of a solubleendoglin-preventive disorder or a soluble endoglin-mediated disordervary depending on the type of compound (e.g., chemical compound, smallmolecule, purified protein, antibody, antisense, RNAi, or nucleic acidvector) and are well known to those skilled in the art or are readilydetermined.

Any of the compositions described herein can be formulated andadministered in a variety of ways, e.g., those routes known for specificindications, including, but not limited to, topically, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraarterially, intralesionally, parenterally,intraventricularly in the brain, or intraocularly. Any of thecompositions described above can be in the form of a pill, tablet,capsule, liquid, or sustained-release tablet for oral administration; ora liquid for intravenous or subcutaneous, systemic administration; or apolymer or other sustained-release vehicle for local administration.

The provided compositions can be administered continuously by infusion,using a constant- or programmable-flow implantable pump, or by periodicinjections. Sustained-release systems can also be used. Administrationcan be continuous or periodic. Semi-permeable, implantable membranedevices are also useful as a means for delivering any of thecompositions of the invention (e.g., anti-soluble endoglin antibodies,soluble endoglin polypeptides, fragments, derivatives, or analogs, orsoluble endoglin nucleic acids). For example, cells that secrete solubleendoglin polypeptide or a fragment of the invention can be encapsulated,and such devices can be implanted into a subject, for example, into aprimary tumor (e.g., a head and neck, a pancreatic, or an esophagealcancer). In another embodiment, any of the above compositions (e.g., asoluble endoglin polypeptide, fragment, derivative, or analog, orsoluble endoglin nucleic acid) is administered locally, e.g., by directinjections, and the injections can be repeated periodically. Such localadministration is particularly useful in the prevention and treatment oflocal metastasis.

Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences (20^(th) edition), Ed.,A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.), inthe form of lyophilized formulations or aqueous solutions. Acceptablecarriers, include saline, or buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments the preservative concentration ranges from 0.1 to 2.0%,typically v/v. Suitable preservatives include those known in thepharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben,and propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant. Preferred surfactants are non-ionic detergents. Preferredsurfactants include Tween 20 and pluronic acid (F68). Suitablesurfactant concentrations are between 0.005 to 0.02%.

In one exemplary in vivo approach, a composition containing solubleendoglin polypeptide of the invention is administered to a subject. Thesoluble endoglin polypeptide can be delivered systemically to thesubject or directly to the target tissue (e.g., to a tumor or a tumorbed following surgical excision of the tumor), in order to prevent orreduce metastasis or to inhibit survival of any remaining tumor ormetastases cells. The dosage required depends on the choice of the routeof administration; the nature of the formulation; the nature of thesubject's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician.

Wide variations in the needed dosage are to be expected in view of thevariety of compositions provided by the invention (e.g., solubleendoglin polypeptides, fragments, derivatives, and analogs, anti-solubleendoglin antibodies, compounds, and soluble endoglin nucleic acids) andthe differing efficiencies of various routes of administration. Forexample, oral administration would be expected to require higher dosagesthan administration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization as is well understood in the art. Administrations can besingle or multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50,100, 150, or more). Encapsulation of the composition (e.g., acomposition containing a soluble endoglin polypeptide, fragment,derivative, antibody, or fragment thereof, or analog of the invention)in a suitable delivery vehicle (e.g., polymeric microparticles,nanoparticle, or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

Alternatively, a composition of the invention can be delivered to theappropriate cells in the subject. For example, a polynucleotidecontaining a nucleic acid sequence encoding a soluble endoglinpolypeptide (as described above) can be directed to any cell in the bodyof the subject. In certain embodiments, expression of the codingsequence can be directed to a tumor or metastases. This can be achievedby, for example, the use of polymeric, biodegradable microparticle,nanoparticle, or microcapsule delivery devices known in the art.

A nucleic acid (e.g., a nucleic acid encoding a soluble endoglinpolypeptide described herein or an antisense oligomer that iscomplementary to a sequence encoding a soluble endoglin polypeptidedescribed herein) can be introduced into the cells by any meansappropriate for the vector employed. Many such methods are well known inthe art (Sambrook et al., supra, and Watson et al., Recombinant DNA,Chapter 12, 2nd edition, Scientific American Books, 1992). Examples ofmethods of gene delivery include liposome-mediated transfection,electroporation, calcium phosphate/DEAE dextran methods, gene gun, andmicroinjection.

The dosage and the timing of administering the compound depends onvarious clinical factors including the overall health of the subject andthe severity of the symptoms. In general, once a solubleendoglin-mediated disorder or a soluble endoglin-preventive disorder, ora propensity to develop a soluble endoglin-mediated disorder or asoluble endoglin-preventive disorder has been detected, any of themethods for administering a composition of the invention can be used totreat or prevent further progression of the disorder. For example,continuous systemic infusion or periodic injection to the site of thedisease of a composition provided by the invention (e.g., a solubleendoglin polypeptide, fragment, derivative, or analog, or a solubleendoglin nucleic acid) can be used to treat or prevent the disorder.Treatment can be continued for a period of time ranging from 1 daythrough the lifetime of the subject, more preferably 1 day to 7 days, 1day to 1 month, 1 to 100 days, 1 day to 6 months, I day to 1 year, 1month to 1 year, 1 day to 5 years, 1 day to 10 years, and mostpreferably 1 to 20 days. For treating subjects, between approximately0.1 mg/kg to 500 mg/kg body weight of the composition (e.g., solubleendoglin polypeptide, fragment, derivative, or analog of the invention)can be administered. A more preferable range is 1 mg/kg to 50 mg/kg bodyweight with the most preferable range being from 1 mg/kg to 25 mg/kgbody weight. Depending upon the half-life of the composition (e.g.,soluble endoglin polypeptide, anti-soluble endoglin antibody, fragment,derivative, or analog of the invention) in the particular subject, thecomposition can be administered between several times per day to once aweek. The methods of the present invention provide for single as well asmultiple administrations, given either simultaneously, or over anextended period of time.

Treatment of a soluble endoglin-mediated disorder or a solubleendoglin-preventive disorder as described herein can be continued for aperiod of time ranging from 1 to 100 days, more preferably 1 to 60 days,and most preferably 1 to 20 days, or two or more (e.g., 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 months or more), or until the completion ofpregnancy (when said subject is pregnant). The dosages will varydepending on each compound and the severity of the disorder, and may betitrated to achieve a steady-state blood serum concentration rangingfrom 1 to 25, 1 to 20 ng/ml, or 1 to 7 ng/ml soluble endoglinpolypeptide.

Where sustained-release administration of composition (e.g., a solubleendoglin polypeptide, fragment, derivative, or analog of the invention)is desired in a formulation with release characteristics suitable forthe treatment of any disease or disorder, microencapsulation iscontemplated. Micro-encapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon-γ (rhIFN-γ), interleukin-2, and MN rgp120 (see, fore.g., Johnson et al., Nat. Med, 2:795-799, 1996; Yasuda, Biomed. Ther.,27:1221-1223, 1993; Hora et al., Bio/Technology, 8:755-758, 1990;Cleland, “Design and Production of Single Immunization Vaccines UsingPolylactide Polyglycolide Microsphere Systems,” in “Vaccine Design: TheSubunit and Adjuvant Approach,” Powell and Newman, Eds., Plenum Press:New York, pp. 439-462, 1995; WO 97/03692; WO 96/40072; WO 96/07399; andU.S. Pat. No. 5,654,010; each incorporated by reference).

The sustained-release formulations may include those developed usingpoly(lactic-co-glycolic acid) (PLGA) polymer. The degradation productsof PLGA, lactic and glycolic acids, can be cleared quickly within thehuman body. Moreover, the degradability of this polymer can be adjustedfrom months to years depending on its molecular weight and composition.See, Lewis, “Controlled release of bioactive agents fromlactide/glycolide polymer,” in M. Chasin and Dr. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, pp. 1-41, 1990).

An anti-soluble endoglin antibody, soluble endoglin polypeptide,fragment, derivative, or analog for use in the present invention mayalso be modified in a way to form a chimeric molecule comprising solubleendoglin polypeptide, fragment, derivative, or analog fused to another,heterologous polypeptide or amino acid sequence, such as an Fc sequence(e.g., a soluble endoglin immunoadhesin), or an additional therapeuticmolecule (e.g., a chemotherapeutic or cytotoxic agent).

The soluble endoglin polypeptide, fragment, derivative, or analog, orthe soluble endoglin nucleic acid of the invention can be packaged aloneor in combination with other therapeutic compounds as a kit (e.g., achemotherapeutic agent, an angiogenesis inhibitor, or ananti-proliferative compound). Non-limiting examples include kits thatcontain, e.g., two pills, a pill, and a powder, a suppository, a liquidin a vial, two topical creams, etc.

The kit can include optional components that aid in the administrationof the unit dose to patients, such as vials for reconstituting powderforms, syringes for injection, customized IV delivery systems, inhalers,etc. Additionally, the unit dose kit can contain instructions forpreparation and administration of the compositions. The kit may bemanufactured as a single use unit dose for one patient, multiple usesfor a particular patient (at a constant dose or in which the individualcompounds may vary in potency as therapy progresses); or the kit maycontain multiple doses suitable for administration to multiple patients(“bulk packaging”). The kit components may be assembled in cartons,blister packs, bottles, tubes, and the like.

The diagnostic methods described herein and methods known in the art canbe used to monitor a soluble endoglin-mediated disorder or a solubleendoglin-preventive disorder during therapy or to determine the dosagesof therapeutic compounds. In one example, a therapeutic composition(e.g., an antibody that specifically binds to the soluble endoglinpolypeptide of the invention) is administered to a subject havingpre-eclampsia and the PAAI is determined during the course of therapy.If the PAAI is less than 20, preferably less than 10, then thetherapeutic dosage is considered to be an effective dosage. In anotherexample, a therapeutic compound is administered and the soluble endoglinanti-angiogenic index is determined during the course of therapy. Inanother example, diagnostic methods that utilize an antibody thatspecifically binds to the C-terminus of the soluble endoglin polypeptide(e.g., specifically binds to the sequence of SEQ ID NO: 2), or afragment thereof (e.g., 3 amino acids, 4 amino acids, 5 amino acids, or6 amino acids) may be used to monitor a soluble endoglin-mediateddisorder or a soluble endoglin-preventive disorder during therapy or todetermine the dosages of therapeutic compounds.

Ex Vivo Therapies

Ex vivo strategies can also be used for the treatment of a solubleendoglin-mediated disorder and a soluble endoglin-preventive disorder.

For the treatment of a soluble endoglin-preventive disorders, desirableex vivo strategies involve transfecting or transducing cells obtainedfrom the subject with a nucleic acid encoding a soluble endoglinpolypeptide or fragment of the invention. The transfected or transducedcells are then returned to the subject. The cells can be any of a widerange of types including, without limitation, hematopoietic cells (e.g.,bone marrow cells, macrophages, monocytes, dendritic cells, T-cells, orB-cells), fibroblasts, epithelial cells, endothelial cells,keratinocytes, or muscle cells. Such cells act as a source of a solubleendoglin polypeptide or fragment for as long as they survive in thesubject. Alternatively, tumor cells (e.g., any of those listed herein),preferably obtained from the subject, but potentially from an individualother than the subject, can be transfected or transformed by a vectorencoding a soluble endoglin polypeptide or fragment of the invention.The tumor cells, preferably treated with an agent (e.g., ionizingirradiation) that ablates their proliferative capacity, are thenintroduced into the patient, where they secrete an exogenous solubleendoglin polypeptide or fragment.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the soluble endoglin polypeptide or fragment of theinvention. These methods are known in the art of molecular biology. Thetransduction step is accomplished by any standard means used for ex vivogene therapy including calcium phosphate, lipofection, electroporation,viral infection, and biolistic gene transfer. Alternatively, liposomesor polymeric microparticles can be used. Cells that have beensuccessfully transduced can then be selected, for example, forexpression of the coding sequence or of a drug resistance gene. Thecells may then be lethally irradiated (if desired) and injected orimplanted into the patient.

For the treatment of soluble endoglin-mediated disorders, desirable exvivo strategies can also involve plasmapheresis using a column or resinthat contains antibodies that specifically bind the soluble endoglinpolypeptides or fragments described herein (e.g., antibodies that bindto the C-terminal sequence of SEQ ID NO: 2), or a fragment thereof(e.g., 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids).In these methods, the blood plasma from a patient having a solubleendoglin-mediated disorder is removed from the subject, passed through acolumn that contains antibodies that specifically bind a solubleendoglin polypeptide of the invention, and returned to the subject. Thistechnique results in the removal of soluble endoglin polypeptide (asdescribed herein) from a subject suffering from a solubleendoglin-mediated disorder.

Subject Monitoring Soluble Endoglin-Mediated Disorders

The disease state or treatment of a subject having a solubleendoglin-mediated disorder as described herein (e.g., pre-eclampsia,eclampsia, or a predisposition to such a disorder) can be monitoredusing the diagnostic methods described herein and the methods known inthe art. For example, elevated soluble endoglin polypeptide present in abodily fluid, such as blood, serum urine, plasma, amniotic fluid, orCSF, can be monitored, wherein elevated levels of soluble endoglinpolypeptide of the invention indicate that the subject has a solubleendoglin-mediated disorder or a predisposition for developing a solubleendoglin-mediated disorder. The soluble endoglin polypeptide describedherein may be monitored in addition to the expression levels of sFlt-1,VEGF, P1GF, TGF-β, or eNOS polypeptide or nucleic acid, or PGI2 (see,diagnostic methods described in WO 08/030,283). Such monitoring may beuseful, for example, in assessing the efficacy of a particular drug in asubject or in assessing disease progression. Therapeutics that decrease(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or even 100%) the expression (protein or mRNA) or a biologicalactivity of a soluble endoglin polypeptide or fragment of the inventionare taken as particularly useful for the treatment of a solubleendoglin-mediated disorder. Non-limiting methods for the monitoring ofsoluble endoglin mediated disorders (e.g., pre-eclampsia and eclampsia)are described in WO 08/030,283 (incorporated by reference). Desirably,the treatment of a subject having a soluble endoglin-mediated disorderresults in at least a 10% decrease (e.g., at least a 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even100% decrease) in the severity of one or more (e.g., 2, 3, 4, 5, or 6)symptoms of a soluble endoglin-mediated disorder. Non-limiting examplesof symptoms of a soluble endoglin-mediated disorder include high bloodpressure, proteinuria, blurred vision, blindness, abdominal pain,excessive bruising, headaches, excessive swelling, excessive weightgain, vaginal bleeding, cramping, decreased fetal movement, malaise,nausea, paresthesia, seizure, intravascular coagulation, reduced fetalsize, fever, chills, sweats, dry cough, muscle pain, and enlargedspleen.

Soluble Endoglin-Preventive Disorders

The disease state or treatment of a subject having a solubleendoglin-preventive disorder (e.g., cancer, cerebral arteriovenousmalformations, cardiovascular disorders, and inflammatory disorders) canbe monitored using the diagnostic methods described herein and themethods known in the art. For example, a subject receiving treatment maybe monitored for an increase (e.g., at least 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, or 80% increase) in the expression of a solubleendoglin polypeptide or nucleic acid of the invention or for an increase(e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%increase) in a biological activity of a soluble endoglin polypeptide. Inone example, the soluble endoglin biological activity includesantagonism of TGF-β signaling. An increase in the expression of asoluble endoglin polypeptide or nucleic acid of the invention, or anincrease in a biological activity of a soluble endoglin polypeptideindicates successful treatment of an individual having a solubleendoglin-preventive disorder. Additionally, a subject receivingtreatment for a soluble endoglin-preventive disorder may also bemonitored using methods known in the art. Desirably, the treatment of asubject having a soluble endoglin-preventive disorder results in atleast a 10% decrease (e.g., at least a 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%decrease) in the severity of one or more (e.g., 2, 3, 4, 5, or 6)symptoms of a soluble endoglin-preventive disorder. Non-limitingexamples of symptoms of a soluble endoglin-preventive disorder includewithout limitation headaches, seizures, intracranial hemorrhage, loss ofconsciousness, nausea, vomiting, incontinence, blurred vision,hemiparesis, aphasia, angina, shortness of breath, heart palpitations,faster heartbeat, weakness or dizziness, sweating, persistent cough,blood-tinged saliva, diarrhea, blood in stool, anemia, breast lump ordischarge, lumps in the testes, change in urination, blood in urine,hoarseness, swollen glands, change in wart or mole, indigestion ordifficulty swallowing, vaginal bleeding or discharge, unexpected weightloss, night sweats, fever, skin color changes, itching in anal orgenital area, non-healing sores, headaches, back pain, pelvic pain, andbloating.

The above symptoms of a soluble endoglin-mediated disorder and a solubleendoglin-preventive disorder can be monitored by a physician and thedosage and administration of one of more therapeutic compounds providedby the present invention may be adjusted based on the evaluation ofthese symptoms by the physician.

Diagnostic Assays and Kits Soluble Endoglin-Mediated Disorders

The invention further provides methods for diagnosing a subject ashaving a soluble endoglin-mediated disorder or a predisposition fordeveloping a soluble endoglin-mediated disorder. A diagnostic testmeasuring soluble endoglin polypeptide having a soluble endoglinbiological activity and containing a sequence that is at least 70%identical (e.g., at least 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to thesequence of SEQ ID NO: 2, or a fragment thereof (e.g., 3 amino acids, 4amino acids, 5 amino acids, or 6 amino acids), optionally furthermeasuring sFlt1 and free P1GF is useful for the detection and monitoringof a person having, or having a predisposition for developing, a solubleendoglin-mediated disorder. One example of an antibody useful for any ofthe diagnostic methods and kits of the invention is an antibody (e.g., apolyclonal antibody) that recognizes an epitope comprising a sequencethat is at least 70% identical (e.g., at least 71%, 72%, 73%, 74%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical) to the sequence of SEQ ID NO: 2, or a fragment thereof (e.g.,3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids). In oneembodiment, the antibody is C-5144 or an antibody that competes withC-5144 for binding to soluble endoglin.

The diagnostic test may further measure the levels of free VEGF, TGF-βfamily members (preferably, TGF-β1 or TGF-β3), free activin-A, BMP2,BMP7, NOS (preferably eNOS), or PGI2, either alone or in any combinationthereof. An alteration in the levels of any of these proteins isdiagnostic of a soluble endoglin-mediated disorder (e.g., pre-eclampsiaor eclampsia) (see, e.g., the diagnostic assays described in WO08/030,283; incorporated by reference). In one example, a decrease inthe levels of free BMP2, BMP7, or activin-A indicates that a subject hasor has a predisposition for developing a soluble endoglin-mediateddisorder (e.g., a pregnancy related hypertensive disorder).

Levels of soluble endoglin polypeptide or a fragment of the invention,either free, bound, or total levels, are measured in a subject sampleand used as an indicator of a soluble endoglin-mediated disorder, whereincreased levels of soluble endoglin polypeptide or a fragment thereofindicate that the subject has a soluble endoglin-mediated disorder orhas a predisposition for developing a soluble endoglin-mediateddisorder. The soluble endoglin polypeptide or fragment can include anyof the soluble endoglin polypeptides or fragments described herein, andmay include degradation products, enzymatic cleavage products of solubleendoglin, and the like. An antibody that specifically binds a solubleendoglin polypeptide or fragment (as described herein) may be used forthe diagnosis of a soluble endoglin-mediated disorder or to identify asubject at risk of developing such a disorder. A variety of protocolsfor measuring an alteration in the expression of such polypeptides areknown, including immunological methods (such as ELISAs and RIAs), andprovide a basis for diagnosing a soluble endoglin-mediated disorder oridentifying a subject at risk of developing a soluble endoglin-mediateddisorder.

Increased levels of a soluble endoglin polypeptide or fragment asdescribed herein are positive indicators of a soluble endoglin-mediateddisorder. For example, if the level of a soluble endoglin polypeptide orfragment of the invention is increased relative to a control reference(e.g., an increase of at least 10%, 20%, 30%, 40%, 60%, 70%, 80%, or 90%or more or 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, or more) orthe level increases over time in one or more samples from a subject,this is considered a positive indicator of a soluble endoglin-mediateddisorder. In one specific example, an increase in soluble endoglinlevels of approximately 2-fold may be diagnostic of pulmonaryhypertension or a propensity to develop pulmonary hypertension.Additionally, any detectable alteration in levels of sFlt-1, VEGF, orP1GF relative to normal levels is indicative of a solubleendoglin-mediated disorder or the propensity to develop a solubleendoglin-mediated disorder (see, e.g., the diagnostic assays describedin WO 08/030,283; incorporated by reference). Normally, circulatingserum concentrations of soluble endoglin polypeptide range from 0-10 or2-7 ng/mL (non-pregnant subjects) and 10-20 ng/mL (pregnant subjects).Elevated serum levels, greater than 15 ng/mL, preferably greater than 20ng/mL, and most preferably greater than 25 ng/mL or more, of solubleendoglin polypeptide is considered a positive indicator of eclampsia orpre-eclampsia. Levels of soluble endoglin ranging from about 7 to about12 ng/ml in sera from non-pregnant subjects is generally an indicator ofprimary and/or secondary pulmonary arterial hypertension.

In one embodiment, the level of soluble endoglin is measured incombination with the level of a sFlt-1, VEGF, or P1GF polypeptide ornucleic acid, or any combination thereof. Methods for the measurement ofsFlt-1, VEGF, and P1GF are described in U.S. Patent ApplicationPublication Numbers 2004/0126828, 2005/0025762, and 2005/0170444 and PCTPublication Numbers WO 2004/008946 and WO 2005/077007 (each incorporatedby reference in their entirety). An increase in the level of sFlt-1polypeptide or nucleic acid level, or a decrease in VEGF and P1GFpolypeptide or nucleic acid levels indicate that the subject has an asoluble endoglin-mediated disorder or has a predisposition fordeveloping a soluble endoglin-mediated disorder. In pregnancy-relatedhypertensive disorders, additional criteria, such as the body mass index(BMI) of the mother and the gestational age of the fetus is alsomeasured and included in the diagnostic metric.

In another embodiment, the level of TGF-β1, TGF-β3, or eNOS polypeptideor nucleic acid is measured in combination with the level of a solubleendoglin or fragment, sFlt-1, VEGF, or P1GF polypeptide or nucleic acidof the invention. Antibodies useful for the measurement of TGF-β1 andTGF-β3 polypeptide levels are commercially available, for example, fromAbeam, Abgent, BD Biosciences Pharmingen, Chemicon, GeneTex, and R&DSystems. The level of PGI₂ can also be used in combination with thelevel of any of the above polypeptides. PGI₂ levels can be determined,for example, using the PGI₂ receptor as a binding molecule in any of thediagnostic assays described above, or using, for example, the urinaryprostacyclin colorimetric ELISA kit (Assay Designs). A decrease in PGI₂levels indicates that the subject has a soluble endoglin-mediateddisorder or has a predisposition for developing a solubleendoglin-mediated disorder. Antibodies useful for the measurement ofeNOS polypeptide levels are commercially available, for example, fromResearch Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BDBiosciences.

In another embodiment, the biological activity of any one or more ofTGF-β1, TGF-β3, or eNOS polypeptide is measured in combination with theexpression level of soluble endoglin of the invention (protein or mRNA)or a biological activity of soluble endoglin polypeptide or fragment,and the expression level of a sFlt-1, VEGF, or P1GF polypeptide. Adecrease in the biological activity of TGF-β1, TGF-β3, or eNOS is apositive indicator of a soluble endoglin-mediated disorder (e.g.,pre-eclampsia or eclampsia). The biological activity can be measured,for example using an assay for enzymatic activity or for the downstreamsignaling activity. In one example, the enzymatic activity of eNOS isdetermined by measuring citrulline conversion and a decrease in theenzymatic activity of eNOS is a positive indicator of a solubleendoglin-mediated disorder (e.g., pre-eclampsia or eclampsia).

Additional metrics may be used to diagnose a person as having a solubleendoglin-mediated disorder. Examples of metrics that may be used includePAM (sFlt-1/VEGF+P1GF); anti-angiogenic index: (sFlt-1+0.25(solubleendoglin polypeptide))/P1GF; (soluble endoglin+sFlt-1)/P1GF; andsFlt-1×soluble endoglin polypeptide or fragment. These metrics aredescribed in WO 04/008946; WO 05/07707; and WO 06/034507 (eachincorporated by reference).

In an example for the diagnosis of pregnancy-related hypertensivedisorder (e.g., pre-eclampsia or eclampsia), the levels of sFlt-1 and asoluble endoglin polypeptide or fragment of the invention in the firstand second trimesters is measured in a subject, and the delta value ofsFlt1×soluble endoglin (sEng) is calculated in each trimester using thefollowing equation: [dproduct=(sFlt1×sEng) in the secondtrimester−(sFlt1×sEng) in the first trimester], where a value greaterthan 0, 1, 2, or more, including fractions thereof (e.g., a positivevalue) is a diagnostic indicator of pre-eclampsia or eclampsia. Apositive value can also be an indicator of pre-term pre-eclampsia. Sucha measurement can be taken on numerous occasions during the first andsecond trimesters and the dproduct can be followed over time. Inaddition, the dproduct of the sFlt-1 level (dsFlt-1) and the sEng level(dsEng) alone can also be calculated between the first and secondtrimesters, where a value greater than 0, 1, 2, or more, includingfractions thereof (e.g., a positive value) for (dsFlt-1) or (dsEng) is adiagnostic indicator of pre-eclampsia or eclampsia. In addition, themetric can further include the level of TGF-1β, TGF-β3, PGI₂, or eNOSpolypeptide. Any of the metrics can further include the BMI of themother or the gestational age of the infant.

Standard methods may be used to measure levels of the soluble endoglinpolypeptides or fragments described herein, free VEGF, free P1GF,sFlt-1, TGF-β1, TGF-β3, PGI₂, or eNOS polypeptide in any bodily fluid,including, but not limited to, urine, serum, plasma, saliva, amnioticfluid, or cerebrospinal fluid. Such methods include immunoassay, ELISA,western blotting using antibodies directed to soluble endoglinpolypeptides or fragments, free VEGF, free P1GF, sFlt-1, TGF-β1, TGF-β3,PGI₂, or eNOS polypeptide and quantitative enzyme immunoassay techniquessuch as those described in Ong et al. (Obstet. Gynecol. 98:608-611,2001) and Su et al. (Obstet. Gynecol., 97:898-904, 2001). ELISA is thepreferred method for measuring levels of a soluble endoglin polypeptideor fragment of the invention, VEGF, P1GF, sFlt-1, TGF-β1, TGF-β3, PGI₂,or eNOS polypeptide. Preferably, a soluble endoglin polypeptide orfragment described herein is measured alone or in combination with anyone or more of the remaining polypeptides. For any of the diagnosticmethods described herein, the soluble endoglin polypeptide is measuredusing an anti-soluble endoglin antibody of the invention including thosethat bind to an epitope that includes SEQ ID NO: 1 or SEQ ID NO: 2 orfragments thereof.

The measurement of any of the nucleic acids or polypeptides describedherein can occur on at least two different occasions and an alterationin the levels as compared to normal reference levels over time can beused as an indicator of a soluble endoglin-mediated disorder or thepropensity to develop such a disorder.

In one example, the level of a soluble endoglin polypeptide or fragment,or a soluble endoglin nucleic acid of the invention present in thebodily fluids of a subject having a soluble endoglin-mediated disorderor having a propensity to develop such a disorder may be increased by aslittle as at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more)relative to levels in a normal control subject or relative to a previoussampling obtained from the same bodily fluid of the same subject. Inanother example, the level of a soluble endoglin polypeptide, fragment,or nucleic acid of the invention in the bodily fluids of a subjecthaving a soluble endoglin-mediated disorder or having a propensity todevelop such a disorder may be altered by as little as at least 5%(e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more) over time from onemeasurement to the next.

The level of sFlt-1, VEGF, or P1GF measured in combination with thelevel of a soluble endoglin polypeptide, fragment, or nucleic acid ofthe invention in the bodily fluids of a subject having a solubleendoglin-mediated disorder or having the propensity to develop such adisorder may be altered by as little as at least 5% (e.g., at least 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, or 99% or more) relative to the level of sFlt-1, VEGF, orP1GF in a normal control. The level of sFlt-1, VEGF, or P1GF measured incombination with the level of a soluble endoglin polypeptide, fragmentor nucleic acid of the invention in the bodily fluids of a subjecthaving a soluble endoglin-mediated disorder or having the propensity todevelop such a disorder may be altered by as little as at least 5%(e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more) over time from onemeasurement to the next.

In one embodiment, a subject sample of a bodily fluid (e.g., urine,blood, plasma, serum, amniotic fluid, or cerebrospinal fluid) iscollected from a subject prior to the onset of symptoms of a solubleendoglin-mediated disorder. In another example, the sample can be atissue or cell collected from the subject prior to the onset of symptomsof a soluble endoglin-mediated disorder. Non-limiting examples oftissues and cells include placental tissue, placental cells, circulatingendothelial cells, and leukocytes such as monocytes. In humans, forexample, maternal blood serum samples are collected from the antecubitalvein of pregnant women during the first, second, or third trimesters ofthe pregnancy. The assay may be carried out during the first trimester,for example, at 4, 6, 8, 10, or 12 weeks, or any interval therein, orduring the second trimester, for example at 14, 16, 18, 20, 22, or 24weeks, or any interval therein. In one example, the assay is carried outbetween 13 and 16 weeks of pregnancy. Such assays may also be conductedat the end of the second trimester or the third trimester, for exampleat 26, 28, 30, 32, 34, 36, or 38 weeks, or any interval therein. It ispreferable that the level of a soluble endoglin polypeptide, fragment,or nucleic acid of the invention and/or any of the additionalpolypeptides described herein be measured twice during this period oftime. For the diagnosis of post-partum pre-eclampsia or eclampsia,assays for a soluble endoglin polypeptide, fragment, or nucleic aciddescribed herein may be carried out postpartum. For the diagnosis of apredisposition to pre-eclampsia or eclampsia, the assay is carried outprior to the onset of pregnancy or prior to the development of symptomsof pre-eclampsia or eclampsia. In one example, for the monitoring andmanagement of therapy, the assay is carried out during the pregnancyafter the diagnosis of pre-eclampsia, and/or during therapy.

In one particular example, serial blood samples can be collected from asubject and the level of a soluble endoglin polypeptide or fragmentand/or any of the additional polypeptides of the invention determined byELISA. In another example, a sample is collected during the secondtrimester and early in the third trimester and in increase in the levelof a soluble endoglin polypeptide or fragment and/or any of the otherpolypeptides of the invention from the first sampling to the next isindicative of pre-eclampsia or eclampsia, or the propensity to developeither.

In veterinary practice, assays may be carried out at any time during thepregnancy, but are, preferably, carried out early in pregnancy, prior tothe onset of pre-eclampsia symptoms. Given that the term of pregnanciesvaries widely between species, the timing of the assay will bedetermined by a veterinarian, but will generally correspond to thetiming of assays during a human pregnancy.

The diagnostic methods described herein can be used individually or incombination with any other diagnostic method described herein or knownin the art for a more accurate diagnosis of the presence of, severityof, or estimated time of onset of a soluble endoglin-mediated disorder.In addition, the diagnostic methods described herein can be used incombination with any other diagnostic methods determined to be usefulfor the accurate diagnosis of the presence of, severity of, or estimatedtime of onset of a soluble endoglin-mediated disorder.

The diagnostic methods described herein can also be used to monitor andmanage a soluble endoglin-mediated disorder in a subject. In oneexample, a therapy is administered until the blood, plasma, or serumsoluble endoglin polypeptide or fragment level is less than 25 ng/ml inpregnant subjects or less than 7-12 ng/ml in sera of subject sufferingfrom PAH, or until the serum soluble endoglin polypeptide or fragmentlevels (or soluble endoglin binding protein, PGI₂, or eNOS level) returnto the baseline level determined before onset of the a solubleendoglin-mediated disorder. In another example, if a subject isdetermined to have an increased level of soluble endoglin polypeptide orfragment of the invention relative to a normal control then the therapycan be administered until the serum P1GF level rises to approximately400 pg/mL or a return to baseline level prior to onset of the solubleendoglin-mediated disorder. In this embodiment, the levels of solubleendoglin polypeptide or fragment described herein, sFlt-1, P1GF, VEGF,soluble endoglin binding protein, PGI₂, eNOS or any and all of these,are measured repeatedly as a method of not only diagnosing disease butmonitoring the treatment and management of the soluble endoglin-mediateddisorder.

Soluble Endoglin-Preventive Disorder

The invention further provides methods for diagnosing a subject ashaving a soluble endoglin-preventive disorder or having a predispositionfor developing a soluble endoglin-preventive disorder. The diagnosticmethods and kits provided by the invention require the measurement of asoluble endoglin polypeptide or fragment as described herein andoptionally further require measuring sFlt1, free P1GF, free VEGF, TGF-βfamily members (preferably, TGF-β1 or TGF-β3), free activin-A, BMP2,BMP7, NOS (preferably eNOS), or PGI2, either alone or in any combinationthereof (wherein increased levels of one or more of these additionalproteins is indicative of a soluble endoglin-preventive disorder). Analteration in the levels of any of these additional proteins isdiagnostic of a soluble endoglin-preventive disorder (e.g., cancer).

Levels of a soluble endoglin polypeptide or fragment described herein,either free, bound, or total levels, are measured in a subject sampleand used as an indicator of a soluble endoglin-preventive disorder,where decreased levels of a soluble endoglin polypeptide or fragmentindicate that the subject has a soluble endoglin-preventive disorder orhas a predisposition for developing a soluble endoglin-preventivedisorder. The soluble endoglin polypeptide or fragment can include anyof the soluble endoglin polypeptides or fragments described herein, andmay include degradation products, enzymatic cleavage products of solubleendoglin, and the like. An antibody that specifically binds a solubleendoglin polypeptide may be used for the diagnosis of a solubleendoglin-preventive disorder or to identify a subject at risk ofdeveloping such a disorder. One example of an antibody useful for themethods of the invention is an antibody (e.g., a polyclonal antibody)that recognizes an epitope comprising a sequence that is at least 70%identical (e.g., at least 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to thesequence of SEQ ID NO: 2, or a fragment thereof (e.g., 3 amino acids, 4amino acids, 5 amino acids, or 6 amino acids).

Decreased levels of a soluble endoglin polypeptide or fragment asdescribed herein or increased levels of TGF-β1 or TGF-β3 are positiveindicators of a soluble endoglin-preventive disorder. For example, ifthe level of soluble endoglin polypeptide or fragment of the inventionis decreased relative to a control reference (e.g., decreased by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%,80%, 90%, or 99% or more relative to a control reference) or decreasesover time in one or more samples from a subject, this is considered apositive indicator of a soluble endoglin-preventive disorder.

As described above, the levels of sFlt-1, VEGF, P1GF, TGF-β1, TGF-β3,eNOS, activin A, BMP2, BMP7, and PGI₂ may be measured using methodsknown in the art. In additional preferred embodiments, the physicalsymptoms of a soluble endoglin-preventive disorder are also determinedby a physician.

Diagnostic Kits

The invention also provides diagnostic kits containing components toperform the diagnostic methods described above. For example, adiagnostic kit can include one or more binding agents (e.g.,polypeptides and the anti-soluble endoglin antibodies of the invention)that specifically bind to the soluble endoglin polypeptides or fragmentdescribed herein and components for detecting, and more preferablyevaluating, binding between the binding agent and the soluble endoglinpolypeptide or fragment.

The diagnostic kits also generally include a label or instructions forthe intended use of the kit components and a reference sample orpurified proteins to be used to establish a standard curve. In oneexample, the kit contains instructions for the use of the kit for thediagnosis of a soluble endoglin-mediated disorder (e.g., pre-eclampsiaor eclampsia) or a soluble endoglin-preventive disorder (e.g., cancer),or instructions for identifying a subject having a propensity to developone of these disorders. In yet another example, the kit containsinstructions for the use of the kit to monitor therapeutic treatment ordosage regimens for the treatment of a soluble endoglin-mediateddisorder or a soluble endoglin-preventive disorder. The diagnostic kitmay also include a label or instructions for the use of the kit todetermine metric values (e.g., the PAAI or the soluble endoglinanti-angiogenesis index) of the subject sample (as described above) andinstructions to compare the metric to a reference sample value. It willbe understood that the reference sample values will depend on theintended use of the kit. For example, the sample can be compared to anormal reference value, wherein an increase in the PAAI, the solubleendoglin anti-angiogenesis index, or the soluble endoglin value isindicative of a soluble endoglin-mediated disorder (e.g., pre-eclampsiaor eclampsia). In another example, the sample can be compared to anormal reference value, wherein a decrease in the PAM, the solubleendoglin anti-angiogenesis index, or the soluble endoglin value isindicative of a soluble endoglin-preventive disorder (e.g., cancer). Inanother example, a kit used for therapeutic monitoring can have areference PAAI, a soluble endoglin anti-angiogenesis index value, or asoluble endoglin value that is indicative of a soluble endoglin-mediateddisorder (e.g., pre-eclampsia or eclampsia), wherein an decrease in thePAAI, the soluble endoglin anti-angiogenesis index value, or the solubleendoglin value of the subject sample relative to the reference samplecan be used to indicate therapeutic efficacy or effective dosages oftherapeutic compounds for the treatment of a soluble endoglin-mediateddisorder. In an additional example, a kit used for therapeuticmonitoring can have a reference PAAI, a soluble endoglinanti-angiogenesis index value, or a soluble endoglin value that isindicative of a soluble endoglin-preventive disorder (e.g., cancer),wherein an increase in the PAM, the soluble endoglin anti-angiogenesisindex value, or the soluble endoglin value of the subject samplerelative to the reference sample can be used to indicate therapeuticefficacy or effective dosages of therapeutic compounds for the treatmentof a soluble endoglin-preventive disorder. A standard curve of levels ofa purified soluble endoglin polypeptide, fragment, or nucleic acid ofthe invention within the normal or positive reference range, dependingon the use of the kit, can also be included.

Screening Assays Compounds for the Treatment of an SolubleEndoglin-Mediated Disorder

As discussed above, the level of a soluble endoglin polypeptide,fragment, or nucleic acid of the invention is increased in a subjecthaving a soluble endoglin-mediated disorder. Soluble endoglincompositions of the invention are useful for the high-throughputlow-cost screening of candidate compounds to identify those moleculesthat modulate the expression of the soluble endoglin polypeptides,fragments, or nucleic acids described herein.

Any number of methods are available for carrying out screening assays toidentify new candidate compounds that alter the expression of a solubleendoglin nucleic acid molecule described herein. Examples are describedin detail in U.S. Patent Application Publication No. 2006/0067937 andPCT Publication No. WO 06/034507 (each incorporated by reference).

In one working example, candidate compounds may be screened for thosethat specifically bind to a soluble endoglin polypeptide, or fragment,described herein. Desirably, the candidate compounds specifically bindto a sequence that is at least 75% identical to the C-terminal sequenceof SEQ ID NO: 2, or a fragment thereof (e.g., 3 amino acids, 4 aminoacids, 5 amino acids, or 6 amino acids). The efficacy of such acandidate compound is dependent upon its ability to interact with such asoluble endoglin polypeptide, or fragment, described herein, or afunctional equivalent thereof. Such an interaction can be readilyassayed using any number of standard binding techniques and functionalassays such as immunoassays or affinity chromatography based assays(e.g., those described in Ausubel et al., supra). In one embodiment, asoluble endoglin polypeptide, or fragment, of the invention isimmobilized and compounds are tested for the ability to bind to theimmobilized soluble endoglin polypeptide, or fragment, using standardaffinity chromatography based assays. Compounds that bind to theimmobilized soluble endoglin polypeptide, or fragment, of the inventioncan then be eluted and purified, and tested further for their ability tobind to the soluble endoglin polypeptide or fragment both in vivo and invitro, or tested for their ability to inhibit a biological activity of asoluble endoglin polypeptide or fragment.

In another example, a candidate compound is tested for its ability todecrease a biological activity of a soluble endoglin polypeptide, orfragment, as described herein. For example, a candidate compound may betested for its ability to decrease the binding of a soluble endoglinpolypeptide, or fragment, to a growth factor, such as TGF-β1, TGF-β3,activin-A, BMP-2, BMP-7, and BMP-9. These binding assays can beperformed in vivo or in vitro using methods known in the art (e.g., see,WO 08/030,283). Additional biological activities of a soluble endoglinpolypeptide, or fragment, described herein can be measured using any ofthe assays for measuring a soluble endoglin biological activitydescribed above or known in the art.

Compounds for the Treatment of an Soluble Endoglin-Preventive Disorder

Compounds capable of increasing the expression levels (protein or mRNA)or biological activity of a soluble endoglin polypeptide of theinvention, useful for the treatment of a soluble endoglin-preventivedisorder, may be identified from large libraries of both natural productor synthetic (or semi-synthetic) extracts, chemical libraries, or frompolypeptide or nucleic acid libraries, according to methods known in theart. Those skilled in the field of drug delivery and development willunderstand that the precise source of test extracts or compounds is notcritical to the screening procedure(s) of the invention.

Any number of methods are available for carrying out screening assays toidentify new candidate compounds that modulate the expression orbiological activity of a soluble endoglin polypeptide, fragment, ornucleic acid of the invention. Examples are described in detail in U.S.Patent Application Publication No. 2006/0067937 and PCT Publication No.WO 06/034507 (incorporated by reference).

The following examples are meant to illustrate the invention. They arenot meant to limit the invention in any way.

EXAMPLES Example 1 Identification and Characterization of Human SolubleEndoglin (HsEng)

Soluble receptors are important mediators that are generated by eitherproteolytic cleavage of membrane-anchored receptors or alternativepre-mRNA splicing, resulting in truncated molecules lacking atransmembrane and intracellular domains. While some of these receptorsact as antagonists by retaining their ability to bind ligands, othersintercalate or dimerize with their cell surface receptor counterparts toeither enhance or impair signal transduction. We have previouslyreported on a 64 kD circulating form of Eng, which we termed solubleendoglin (sEng) and found that this protein was elevated 10-20-fold inthe sera of patients with pre-eclampsia. Peptide analysis of purifiedsEng by mass spectrometry revealed sequences belonging to parts of theN-terminal extracellular region of the full-length transmembraneglycoprotein. Not knowing the exact amino acid sequence of this form ofsEng, we used a recombinant Eng (rEng) protein, which consisted of theentire extracellular region of cell surface Eng (amino acids 25-586) totest its effects on endothelial cells. Using this approach, wedemonstrated that rEng induced endothelial dysfunction by interferingwith the ability of TGF-β1 to bind to its receptors and to regulateendothelial nitric oxide synthase (eNOS) activity. Taken together, rEngdisplayed marked anti-angiogenic properties and contributed to thepathogenesis of pre-eclampsia. However, despite numerous attempts, theputative C-terminal cleavage site of Eng was never identified andtherefore the true identity and perhaps function of the 64 kD sEngprotein still remains unknown.

We have discovered a previously unrecognized alternatively spliced humanEng mRNA transcript, which contains a unique 3′ sequence and UTR (FIG.2A). This novel Eng mRNA was found using total placental RNA isolatedfrom patients with pre-eclampsia and a modified 5′-3′ rapidamplification of cDNA ends (RACE) method.

Cell surface Eng consists of 14 exons spread over 40 kb on humanchromosome 9. The HsEng mRNA transcript is generated by exon extensionwhereby exon 9b is “extended” by 372 bases into intron 10 (FIG. 2A). Theresulting novel 1683-base Eng mRNA transcript preserves the same ATGstart site as the full-length form of Eng and harbors a stop codon atposition 1333 and 351 bp of 3′ UTR giving rise to a predicted 444-aminoacid protein (FIG. 2A). Given that this transcript encodes the samesignal peptide, but not the transmembrane region of full-length Eng,this suggests that this novel mRNA encodes a secreted protein. Ourquantifications of HsEng mRNA relative to 18S RNA levels using Taqmanprimer/probe sets, suggest that the HsEng transcript is expressed athigh levels in human lung, placenta, uterus and testes (FIG. 2B). Wehave also recently cloned the mouse sEng homolog (MsEng), which isgenerated similarly to HsEng, and found that this transcript is alsomost abundant in the lung. These findings suggest that the regulation ofthese transcripts is conserved across species and underscore thepotential importance of soluble endoglin in the lung.

Overexpression of sEng cDNA in bovine aortic endothelial cells gave riseto a stable protein in the conditioned media with an apparent molecularweight of ˜64 kD under both non-reducing and reducing conditions (FIG.3C). The detection of this specific band supported our notion that ournewly cloned sEng mRNA likely encodes the ˜64 kD protein previouslyisolated previously by our group in the sera of patients withpreeclampsia. While a relatively small portion of sEng was also detectedas a ˜125 kD dimer, these results strongly suggest that sEng existspredominantly as a monomer.

Materials and Methods

3′RACE.

RNA of placentae from patients diagnosed with pre-eclampsia was isolatedwith Trizol (Invitrogen) according to guidelines provided by themanufacturer. Five micrograms of total RNA was reverse transcribed withSupercript III reverse transcriptase and oligo dT primer supplied in theGeneracer 5′-3′ RACE kit (Invitrogen). The cDNA was amplified using thegene-specific primer, 5% atggaccgeggcacgctecctctg-3′ and the 3′ primer(5′-gctgtcaacgatacgctacgtaacg-3′ supplied in the Generacer Kit) with thefollowing PCR cycling parameters: 5 minutes 95° C. denaturation, 1minute 65° C. annealing and 1 minute 72° C. extension for 40 cycles. ThePCR product was run on a 1.2% agarose TAE gel and bands of interest werecut out, purified and subcloned into pCRII-TOPO (Invitrogen).

Human sEng mRNA Expression.

Relative levels of HsEng mRNA were determined by real-time quantitativeRT-PCR (TaqMan protocol). TaqMan analysis was performed using an ABIPrism 7700 sequence detection system (Applied Biosystems). The PCRprimers and TaqMan probe specific for HsEng mRNA were designed usingPrimer Express software 1.5. Primer and probe sequences were as follows:Forward primer, 5′-TGGTCAGCAATGAGGTGATCA-3′ (SEQ ID NO: 5); Reverseprimer, 5′-ACCGTCCATCTCACCCGAA-3′ (SEQ ID NO: 6); TaqMan Probe5′-FAM-CAGTTTCCCGTCAGGCTCACCACC-TAMRA-3′ (SEQ ID NO: 7). Eukaryotic 18SrRNA TaqMan PDAR (predeveloped TaqMan assay reagents) Endogenous Controlreagent mix (Applied Biosystems) was used to amplify 18S rRNA as aninternal control, according to the manufacturer's protocol.

Overexpression of Human sEng Protein.

The HsEng coding sequence (bases 1-1335) was subcloned downstream of thecytomegalovirus (CMV) promoter in the pcDNA-Hygro vector (Invitrogen).Bovine aortic endothelial cells (BECs) were grown in 6-well plates andtransiently transfected with either the pcDNA-Hygro vector (pc-Empty) orthat containing HsEng (pc-HsEng) using Effectene (Qiagen) according tothe manufacturer's guidelines. After 48 hrs, the condition media andcells were harvested for analysis by IP-Western blot. The conditionmedia was spun down at 1500 rpm for 5 minutes to remove cellularcontaminants followed by another spin at 14000 rpm for 15 minutes. Cellswere harvested as previously described (Toporsian et al. Circ Res 2005).The samples were precleared with protein G (GE Healthcare) andimmunoprecipitated with the human endoglin monoclonal antibody, P4A4(1:200 dilution, Santa Cruz). One quarter of the immunoprecipitates(IPs) was separated by SDS-PAGE under non-reducing or reducingconditions, transferred onto nitrocellulose and blotted with a separateEng antibody (T-20, 1:200 dilution, Santa Cruz) directed at theN-terminus of the protein and Hrp-conjugated anti-mouse or -rabbitsecondary antibodies. Bands were visualized by chemiluminence.

Example 2 Affinity Purified sEng-Specific Antibody

Extension of exon 9 into intron 10 gives rise to a novel C-terminus thatis unique to sEng (see FIG. 2A). This sequence was therefore used as animmunogen to generate an affinity purified rabbit polyclonal antibody,which we have named C-5144. This antibody can specifically detect sEngin sera of patients with pre-eclampsia and should not recognize therecombinant (rEng) or transmembrane form of Eng (FIG. 3D) as the HsEngantigenic site is not present in the latter forms of the protein.Indeed, we were able to detect increased levels of HsEng in the sera ofpatients with pre-eclampsia suggesting that the sEng protein generallydetected in normal and preeclamptic sera harbors the unique C-terminalsequence generated as a result of alternative pre-mRNA splicing. Webelieve that the HsEng described herein and the 64 kD protein previouslydetected in this disease is one and the same (FIG. 3D).

Materials and Methods

Generation of Affinity Purified sEng-Specific Antibody.

The sEng-specific peptide, -VRWTVTC- (SEQ ID NO: 2), was synthesized andinjected into rabbits by personnel at Open Biosystems, a subsidiary ofThermoFisher. Crude sera were analyzed for their ability to detect HsEngand selected for affinity purification. The affinity-purified rabbitpolyclonal antibody, is referred to as C-5144. Sera from normal andpatients with preeclampsia were spun at 14000 g for 15 minutes and 50 mLwas immunoprecipitated with a human endoglin monoclonal antibody, P4A4(1:200 dilution, Santa Cruz) as previously described (Toporsian et al.Circ. Res. 96:684-692 (2005), Venkatesha et at. Nat. Med. 12:642-649(2006)). One quarter of the IPs was separated by SDS-PAGE undernon-reducing conditions, transferred onto nitrocellulose and blottedwith C-5144 (1:500) and Hrp-conjugated anti-rabbit secondary antibody.Bands were visualized by chemiluminence.

Example 3 Elevated HsEng Levels in Sera of PAH Patients

We hypothesized that the chronic and local production of HsEng in thelung may account for the manifestation of PAH in individualsirrespective of any germline mutations in genes encoding BMPR2 or ALK1.Given the importance of these receptors in endothelial cell survival andthe potential perturbations of their activities by HsEng either directlyor via its interaction with cell-surface endoglin, we reasoned thatincreased HsEng within the alveolar wall and surroundingmicrovasculature might be a critical event leading to the observed lossof capillaries, alveolar wall thickening and muscularization ofarterioles in PAH. We have found significantly elevated HsEng levels insera of patients with PAH (FIG. 4). Of note, HsEng detected in theseserum samples had the same apparent molecular weight as our positivecontrol, which consisted of the conditioned medium of HEK293T cells thatwere transfected with HsEng cDNA.

Materials and Methods

Sera from normal and patients with pulmonary arterial hypertension (PAH)were spun at 14000 g for 15 minutes and 50 mL was immunoprecipitatedwith a human endoglin monoclonal antibody, P4A4 (1:200 dilution, SantaCruz) as previously described (Toporsian et. al. Circ. Res. 2005,Venkatesha Nat. Med. 2006). One quarter of the IPs was separated bySDS-PAGE under non-reducing conditions, transferred onto nitrocelluloseand blotted with a separate Eng antibody (AF1097, 1:1000, R&D systems)directed at the N-terminus of the protein and Hrp-conjugated anti-goatsecondary antibodies. Bands were visualized by chemiluminence andquantified by densitometry.

Example 4 HsEng Induces Signs of PAH in Rodents

We tested whether or not HsEng is able to induce signs of PAH inrodents. We generated an adenovirus carrying the HsEng cDNA sequencedownstream of a cytomegalovirus (CMV) promoter (Ad-HsEng) and inoculatedmice with either Ad-HsEng or Ad-Empty by intra-tracheal nebulization.These mice were then exposed to hypoxia (12% O₂) for a period of 3 or 6weeks. This route of adenovirus delivery was chosen to specificallytarget the lung and avoid secondary effects such as widespread systemicendothelial dysfunction resulting from intravenous injections, which canpotentially influence our assessment of cardiopulmonary hemodynamics andhistology. Mice exposed to chronic hypoxia after inoculation withAd-HsEng, showed signs of accelerated disease progression includingsignificantly elevated RVSP and RV hypertrophy compared to control micetreated with Ad-Empty and subsequently exposed to hypoxia (FIGS. 5A, B).Interestingly, prolonged exposure to hypoxia (6 weeks) led to asignificant drop in RVSP in the Ad-HsEng-treated group despite evidenceof progressive RV hypertrophy (FIGS. 5A, B). This observation wasassociated with a significant decrease in the RV delta pressure/deltatime (dp/dt) value and RV contractility index in the Ad-HsEng-treatedgroup exposed to hypoxia for 6 weeks (FIGS. 5C, D). The gross physicalbehavior of these mice appeared more sluggish than that seen in mice inthe control (Ad-Empty) group. Control mice exposed to hypoxia for 3 and6 weeks displayed a gradual increase in RV pressure and hypertrophy,demonstrating a normal response to chronic hypoxia.

Example 5 C-terminal Tail is Essential for HsEng Expression

cDNA constructs as described below were used for expression of the HsEngprotein. As shown in FIG. 6, cells transfected with pc-HsEng (whichexpresses amino acids 1-444 of soluble endoglin as shown in FIGS. 1 and3B) produced a stable and glycosylated 64 kD protein in the conditionmedium. A prominent band at ˜50 kD was present in the corresponding cellextracts and likely represents the unglycosylated HsEng protein(expected: 49 kD). Cells transfected with HsEng-437 (which lacks the 7amino acid C-terminal tail) did not give rise to a 64 kD protein in thecondition media, while a faint band was observed in the correspondingcell extracts at ˜50 kD (unglycosylated protein). The 7 amino acidC-terminal sequence in HsEng-437 is necessary to enable the productionof a properly folded, stable and secreted protein.

These findings suggest that an antibody, small molecule inhibitor orsmall inhibitory siRNA/miRNA that can disrupt the apparentprotein-stabilizing role or expression of the 7aa c-terminal peptide canbe potentially useful in altering the half-life and levels of HsEng inHsEng-dependent diseases. Previous reports have suggested the presenceof a proteolytic cleavage site at position 437aa of cell surfaceendoglin (FIGS. 2A and 3B) that can potentially yield a truncated formof the protein. However, the expression of Endo-437 was very transientand is consistent with our current findings.

Materials and Methods

Using the pc-HsEng construct, Val438 was changed to the stop codon, UAG,by standard PCR-based site-directed mutagenesis using forward(5′-ctcatcaccacagcggtagagatggacagtcacg-3′ (SEQ ID NO: 8)) and reverse(5′-cgtgactgtccatctctaccgctgtggtgatgag-3′ (SEQ ID NO: 9)) using theQuickchange protocol (Stratagene). The clone containing the specificmutation was sequenced and the mutated HsEng-437 cDNA insert was cut outby restriction digest and subcloned into a aliquot of the pcDNA-Hygrovector. The pc-empty, pc-HsEng and pc-HsEng-437 constructs weretransiently transfected into human kidney epitheial (HEK) 293T cellsusing the transfection reagent, Fugene (Roche) according to themanufacturer's guidelines. After 48 hrs, the condition media and cellswere harvested for analysis by IP-Western blot. The condition media wasspun down at 1500 rpm for 5 minutes to remove cellular contaminantsfollowed by another spin at 14000 rpm for 15 minutes. Cells wereharvested as previously described (Toporsian et al. Circ Res 2005,supra). The samples were precleared with protein G (GE Healthcare) andimmunoprecipitated with the human endoglin monoclonal antibody, P4A4(1:200 dilution, Santa Cruz). One quarter of the immunoprecipitates(IPs) was separated by SDS-PAGE under non-reducing conditions,transferred onto nitrocellulose and blotted with a separate Eng antibody(AF1097, 1:1000, R&D systems) directed at the N-terminus of the proteinand Hrp-conjugated anti-goat secondary antibodies. Bands were visualizedby chemiluminence.

Example 6 Post-Translational Modification of HsEng

The dimerization of certain secreted or cell surface proteins canprotect them from digestion by proteases, thereby prolonging theirhalf-life and steady-state levels. We have found that the overexpressionof HsEng cDNA in bovine aortic endothelial cells (BECs), predominantlygives rise to a monomeric protein although some dimers could also bedetected. S-nitrosylation of cysteines has quickly emerged as animportant post-translational modification that can have profound effectson protein structure and function. This modification occurs when acysteine thiol reacts with NO. in the presence of an electron acceptorto form an S—NO bond. As shown in FIG. 7, expression of HsEng in HEK293Tcells that do not normally express eNOS (enzyme that produces NO.),resulted in equal amounts of dimeric and monomeric HsEng (left panel).Ex vivo treatment of HsEng produced in HEK293T cells with the NO donor,GSNO, resulted in the S-nitrosylation of HsEng. S-nitrosylation of HsEngreduced the dimer/monomer ratio of this protein and may potentiallyimpact its function.

Materials and Methods

The pc-empty or pc-HsEng constructs were transiently transfected intohuman kidney epitheial (HEK) 293T cells using the transfection reagent,Fugene (Roche) according to the manufacturer's guidelines. After 48 hrs,the condition media were harvested, concentrated using 50K columns(Millipore) and immediately processed for immunoprecipitation asdescribed below. In a separate set, concentrates were treated with theNO donor, GSNO (10 mM) for 1 hour and subjected to biotin-switch usingthe S-Nitrosylated Protein Detection Kit (Cayman Chemical) according tothe manufacturer's guidelines. The biotinylated proteins were enrichedusing M-280 streptavidin dynabeads (Invitrogen protocol), preclearedwith protein G (GE Healthcare) and immunoprecipitated with the humanendoglin monoclonal antibody, P4A4 (1:200 dilution, Santa Cruz). Onequarter of the immunoprecipitates (IPs) was separated by SDS-PAGE undernon-reducing conditions, transferred onto nitrocellulose and blottedwith a separate Eng antibody (AF1097, 1:1000, R&D systems) directed atthe N-terminus of the protein and Hrp-conjugated anti-goat secondaryantibodies. Bands were visualized by chemiluminence and quantified bydensitometry.

Example 7 Generation of pTRE-HsEng-HPRT Mice

We generated a tetracycline-inducible transgenic mouse by placing HsEngcDNA downstream of the minimal Tet-Responsive Element (TRE)-tightpromoter and targeted this construct into the mouse hypoxanthine-guaninephosphoribosyl transferase (HPRT) locus. The HPRT locus is locatedwithin the X chromosome and has two important advantages for thetargeting of transgenes. First, we have parent embryonic stem (ES,Ola/129-derived; agouti coat color) cells lacking a functional Hprtgene, which could be reconstituted by homologous recombination andselected in medium containing hypoxanthine, aminopterin, and thymidine(HAT). Second, the Hprt gene is ubiquitously expressed and the locus istranscriptionally favorable and free of the constraints of higher ordergene regulation. ES cells were electroporated with linearized targetingvector and recombinant ES cells were selected in HAT medium, genotypedand chosen for injection into C57BL/6-derived blastocysts to generatemale agouti mice displaying 80-100% chimerism (FIG. 8A). Mouse chimeraswere mated with C57BL/6 female mice to generate F1 progeny. F1 femalemice displaying agouti coat color are expected to have germlinetransmission of the transgenic X chromosome. Indeed, genotyping of thesemice using PCR primers yielded a 331bp amplicon encompassing the distalend of pTRE-tight promoter and proximal end of the HsEng sequence (FIG.8D). We currently have female F1 mice (pTRE-HsEng-HPRT), which have beenbackcrossed with C57BL/6 males and given birth to pups (N1 generation).These mice will be an excellent resource in understanding the role ofHsEng in various organs by crossing them with commercially availabletransgenic mice capable of organ-specific Dox-inducible expression.

In order to more closely replicate the adenovirus aerosolizationexperiments that have yielded promising preliminary results (FIG. 5A-Dabove), HsEng expression can be specifically targeted in lung bronchialand alveolar type II epithelial cells. To this end, female N1 progenycarrying the pTRE-HsEng transgene were crossed with male homozygousCC10-rtTA mice carrying the reverse tetracycline-controlledtransactivator (rtTA) protein under the control of the ratsecretagloblin (Scgbla1, family 1A, member 1) gene promoter (FIGS. 8B,C). Using this breeding strategy, all progeny will be hemizygous forCC10-rtTA while 50% of male mice will be pTRE-HsEng/CCSP-rtTAbitransgenic and therefore capable of inducing HsEng expression inbronchial and type II lung epithelial cells when CC10-rtTA is expressedupon exposure to Dox, (1 mg/mL, drinking water) (FIG. 8D, showing 4bitransgenic mice). Of note, rtTA-induced expression of a luciferasereporter under the regulation of a TRE promoter has been detected asearly as embryonic day 12.5 in embryos from pregnant females treatedwith Dox (REF). Basal luciferase activity was negligible (<200 RLU) andmaximal activity (>7000 RLU) was reached within 16 hours when exposed toDox (REF). In all experiments, male mice can be used to circumventpotential silencing of our transgene, if it is inherited on an Xchromosome destined for inactivation in female mice.

Preliminary studies indicate that these mice develop signs of PAH. Thesemice can also be tested with the HsEng-specific (C-5144) antibody todetermine inhibition of the onset and progression of PAH in these mice,lending additional support to the therapeutic use of the anti-HsEngantibodies.

Example 7 TNF-α Increases HsEng Protein Expression from EndothelialCells

The effects of TNF-α on HsEng expression were tested using human lungmicrovascular endothelial cells. As shown in FIGS. 10A and B, treatmentof HMVEC-L with TNF-α does not induce any changes in HsEng mRNA levels(FIG. 9A), but increases HsEng protein levels in the condition media(FIG. 9B).

Materials and Methods

Human lung microvascular endothelial cells (HMVEC-L) were grown toconfluence and treated with or without TNF α (50 ng/mL) for 24 hours.Cells were harvested and both RNA and proteins were isolated aspreviously described.

HsEng mRNA Expression.

Relative levels of HsEng mRNA were determined by real-time quantitativeRT-PCR (TaqMan protocol). TaqMan analysis was performed using an ABIPrism 7700 sequence detection system (Applied Biosystems). The PCRprimers and TaqMan probe specific for HsEng mRNA were designed usingPrimer Express software 1.5. Primer and probe sequences were as follows:Forward primer, 5′-TGGTCAGCAATGAGGTGATCA-3′ (SEQ ID NO: 10); Reverseprimer, 5′-ACCGTCCATCTCACCCGAA-3′ (SEQ ID NO: 11); TaqMan Probe5′-FAM-CAGTTTCCCGTCAGGCTCACCACC-TAMRA-' (SEQ ID NO: 12). Eukaryotic 18SrRNA TaqMan PDAR (predeveloped TaqMan assay reagents) Endogenous Controlreagent mix (Applied Biosystems) was used to amplify 18S rRNA as aninternal control, according to the manufacturer's protocol.

HsEng Protein Expression.

The condition media was spun down at 1500 rpm for 5 minutes to removecellular contaminants followed by another spin at 14000 rpm for 15minutes. Cells were harvested as previously described (Toporsian et al.Circ Res 2005). The samples were precleared with protein G (GEHealthcare) and immunoprecipitated with the human endoglin monoclonalantibody, P4A4 (1:200 dilution, Santa Cruz). One quarter of theimmunoprecipitates (IPs) was separated by SDS-PAGE under non-reducingconditions, transferred onto nitrocellulose and blotted with a separateEng antibody (AF1097, 1:1000, R&D systems) directed at the N-terminus ofthe protein and Hrp-conjugated anti-goat secondary antibodies. Bandswere visualized by chemiluminence.

Other Embodiments

All publications, patents, and patent application publications cited inthis specification are incorporated herein by reference as if eachindividual publication or patent were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. An isolated soluble endoglin polypeptide, whereinsaid soluble endoglin polypeptide comprises no more than 444 continguousamino acids, comprises, within said continguous amino acids, an aminoacid a sequence having at least 85% sequence identity to VRWTVTC (SEQ IDNO: 2), and has a soluble endoglin biological activity.
 2. (canceled) 3.The isolated soluble endoglin polypeptide of claim 2, wherein saidsoluble endoglin polypeptide comprises the sequence of SEQ ID NO:
 2. 4.(canceled)
 5. The isolated soluble endoglin polypeptide of claim 1,wherein the soluble endoglin polypeptide comprises the amino acidsequence of SEQ ID NO:
 3. 6.-11. (canceled)
 12. The isolated solubleendoglin polypeptide of claim 1, wherein said soluble endoglinpolypeptide comprises one or more modifications selected from the groupconsisting of: acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, S-nitrosylation,disulfide bond formation, demethylation, formation of cysteine,formation of sulfonic, sulfenic, or sulfinic acid, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation,GPI-anchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids, ubiquitination, D-isomerization, derivitization of watersoluble polymers, addition of a detectable label, and conjugation ofanother protein or therapeutic compound.
 13. An isolated antibody, orantibody binding fragment thereof, that specifically binds to a solubleendoglin polypeptide, where the antibody, or antibody-binding fragment,binds to an epitope comprising a sequence that is at least 85% identicalto VRWTVTC (SEQ ID NO: 2), or a fragment thereof.
 14. The antibody, orantibody binding fragment of claim 13, wherein the antibody orantibody-binding fragment binds to an epitope comprising SEQ ID NO: 2.15. The antibody, or antibody binding fragment of claim 13, wherein theantibody or antibody-binding fragment competes with the antibody C-5144for binding to soluble endoglin. 16.-20. (canceled)
 21. An isolatednucleic acid molecule comprising a nucleic acid sequence having at least95% sequence identity to the sequence of SEQ ID NO: 4, wherein saidnucleic acid sequence encodes the polypeptide of claim
 1. 22. Theisolated nucleic acid molecule of claim 21, wherein the nucleic acidcomprises a sequence that encodes the amino acid sequence of SEQ ID NO:2.
 23. (canceled)
 24. An isolated inhibitory nucleic acid moleculecomprising at least one strand that is complementary to a nucleic acidsequence that encodes an amino acid sequence that is at least 85%identical to VRWTVTC (SEQ ID NO: 2), and wherein the inhibitory nucleicacid molecule reduces or inhibits the expression of a biologicalactivity of a soluble endoglin.
 25. The isolated inhibitory nucleic acidmolecule of claim 24, wherein the inhibitory nucleic acid molecule hasat least one strand that is complementary to a nucleic acid sequencethat encodes the amino acid sequence of SEQ ID NO:
 2. 26. (canceled) 27.The isolated inhibitory nucleic acid molecule of claim 24, wherein theinhibitor nucleic acid molecule is a double-stranded RNA or asingle-stranded DNA.
 28. The isolated inhibitory nucleic acid moleculeof claim 27, wherein the double-stranded RNA is a small interfering RNA(siRNA).
 29. The isolated inhibitory nucleic acid molecule of claim 28,wherein the siRNA is between 17 to 25 nucleotides in length. 30.(canceled)
 31. A method of treating or decreasing the likelihood ofdeveloping a soluble endoglin-mediated disorder in a subject, comprisingthe step of administering to said subject an agent capable of reducingthe expression or biological activity of a soluble endoglin polypeptide,wherein said polypeptide comprises a sequence that is at least 85%identical to VRWTVTC (SEQ ID NO: 2). 32.-34. (canceled)
 35. The methodof claim 31, wherein the agent is the isolated antibody or antibodybinding fragment thereof of claim
 13. 36.-40. (canceled)
 41. The methodof claim 31, wherein the agent is the inhibitory nucleic acid moleculeof claim
 24. 42.-51. (canceled)
 52. A method of treating or decreasingthe likelihood of developing a soluble endoglin-preventive disorder in asubject, comprising the step of administering to the subject theisolated soluble endoglin polypeptide of claim
 1. 53.-66. (canceled) 67.A method of diagnosing a subject as having, or having the predispositionto develop, a soluble endoglin-mediated disorder or a solubleendoglin-preventive disorder, said method comprising the steps of: (a)measuring the level of the soluble endoglin polypeptide of claim 1 in asample from a subject; (b) measuring the level of the soluble endoglinpolypeptide in a control sample; and (c) comparing the level of thesoluble endoglin polypeptide in the subject to the level of the solubleendoglin polypeptide in the control sample; wherein an increase in thelevel of the soluble endoglin polypeptide in the subject as compared tothe level of the soluble endoglin polypeptide in the control samplediagnoses said subject as having, or having a disposition fordeveloping, a soluble endoglin-mediated disorder, or wherein a decreasein the level of the soluble endoglin polypeptide in the subject ascompared to the level of the soluble endoglin polypeptide in the controlsample diagnosis said subject as having, or having a disposition fordeveloping, a soluble endoglin-preventive disorder. 68.-75. (canceled)76. A kit comprising: (a) An agent selected from: i) the isolatedantibody or antibody binding fragment thereof of claim 13; or ii) theisolated soluble endoglin polypeptide of claim 1; and (b) instructionsfor administering said antibody or antibody binding fragment thereof toa subject having a soluble endoglin-mediated disorder or instructionsfor administering said isolated soluble endoglin polypeptide to asubject having a soluble endoglin-preventive disorder. 77.-85.(canceled)